Plant Physiology and Biochemistry 57 (2012) 254e260

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Plant Physiology and Biochemistry

journal homepage: www.elsevier.com/locate/plaphy

Research article

Phytostabilization of nickel by the zinc and cadmium hyperaccumulator Solanum

nigrum L. Are metallothioneins involved?
Pedro Ferraz a, b, Fernanda Fidalgo a, b, Agostinho Almeida c, Jorge Teixeira a, b, *
a

BioFIGeCenter for Biodiversity, Functional & Integrative Genomics, Portugal

Departamento de Biologia, Faculdade de Cincias, Universidade do Porto, Ed. FC4, Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal
c
REQUIMTE, Departamento de Cincias Qumicas, Faculdade de Farmcia, Universidade do Porto, Rua Anbal Cunha, 164, 4090-030 Porto, Portugal
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 1 March 2012
Accepted 29 May 2012
Available online 6 June 2012

Some heavy metals (HM) are highly reactive and consequently can be toxic to living cells when present at
high levels. Consequently, strategies for reducing HM toxicity in the environmental must be undertaken.
This work focused on evaluating the Nickel (Ni) accumulation potential of the hyperaccumulator Solanum
nigrum L., and the participation of metallothioneins (MT) in the plant Ni homeostasis. Metallothioneins
(MT) are gene-encoded metal chelators that participate in the transport, sequestration and storage of
metals. After different periods of exposure to different Ni concentrations, plant biometric and
biochemical parameters were accessed to determine the effects caused by this pollutant. Semiquantitative RT-PCR reactions were performed to investigate the specic accumulation of MT-related
transcripts throughout the plant and in response to Ni exposure. The data obtained revealed that Ni
induced toxicity symptoms and accumulated mostly in roots, where it caused membrane damage in the
shock-treated plants, with a parallel increase of free proline content, suggesting that proline participates
in protecting root cells from oxidative stress. The MT-specic mRNA accumulation analysis showed that
MT2a- and MT2d-encoding genes are constitutively active, that Ni stimulated their transcript accumulation, and also that Ni induced the de novo accumulation of MT2c- and MT3-related transcripts in shoots,
exerting no inuence on MT1 mRNA accumulation. These results strongly suggest the involvement of
MT2a, MT2c, MT2d and MT3 in S. nigrum Ni homeostasis and detoxication, this way contributing to the
clarication of the roles the various types of MTs play in metal homeostasis and detoxication in plants.
2012 Elsevier Masson SAS. All rights reserved.

Keywords:
Black nightshade
Gene expression
Heavy metal homeostasis
Metallothioneins
Nickel
Phytoremediation

1. Introduction
The environmental pollution caused by heavy metals (HM) is,
nowadays, a major ecological problem with disastrous future
consequences to our planet. The origin of these pollutants in the
environment comes primarily from the release of untreated
industrial waste efuents. The ecological unconsciousness
combined with the lack of legislation in many countries has led to
the accumulation of these metals in soils and watercourses,
affecting all living organisms, from bacteria to animals, including
Abbreviations: FW, fresh weight; HM, heavy metals; MDA, malondialdehyde;
MT, metallothioneins; ROS, Reactive Oxygen Species; RT-PCR, Transcriptase reaction
coupled to the polymerase chain reaction.
* Corresponding author. Departamento de Biologia, Faculdade de Cincias, Universidade do Porto, Ed. FC4, Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal.
Tel.: 351 220402701; fax: 351 220402709.
E-mail addresses: ferraz87@gmail.com (P. Ferraz), fdalgo@fc.up.pt (F. Fidalgo),
aalmeida@ff.up.pt (A. Almeida), agteixei@fc.up.pt, jorgeg.teixeira@gmail.com
(J. Teixeira).
0981-9428/$ e see front matter 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.plaphy.2012.05.025

humans [1]. An example of such pollutants is nickel (Ni), an

element that is essential to some living beings, including plants, but
that could be an environmental contaminant when present in high
concentrations [2].
Ni is the twenty-second most abundant element in the earths
crust, where it occurs in igneous rocks as a free metal or together
with iron [3]. Ni is released into the environment mainly from
anthropogenic activities, such as metal mining, smelting, fossil fuel
burning, vehicle emissions, industrial and municipal wastes and
fertilizer applications [2]. Ni is essential for higher plants in low
concentrations, and its uptake and transport is involved in some
plant physiological processes. The uptake of Ni in plants is carried
out mainly by root systems via passive diffusion (cation transport
system) and active transport, using the magnesium (Mg) ion
transport system, due to the similar charge/size ratio of these two
metal ions, or by high-afnity nickel transport proteins [2].
Although Ni is an essential mineral nutrient for plant growth,
excess Ni has been reported to cause toxicity symptoms including
retardation of germination, growth inhibition, leaf necrosis and

P. Ferraz et al. / Plant Physiology and Biochemistry 57 (2012) 254e260

chlorosis, and disruption of photosynthesis [2,4,5]. Some studies

suggest that Ni toxicity in plants is associated with the interference
of this metal with the uptake and transport of other essential metal
ions, such as iron, copper and zinc, and with the induction of
oxidative stress, which leads to DNA damage and alterations in cell
membranes [2].
Plants have developed several antioxidant mechanisms, both
enzymatic and non-enzymatic, to prevent the damage caused by
the overproduction of ROS. Concerning non-enzymatic mechanisms, the accumulation of soluble proline is recognized as having
an important protective function against heavy metal stress, being
reported to act as a radical scavenger or involved in metal chelation
[6]. In addition, the lipid-soluble antioxidants carotenoids play
a multitude of functions in plant metabolism including oxidative
stress tolerance [7]. Thus, the quantication of membrane damage,
carotenoids and free proline levels in plants exposed to high HM
concentrations serve as indicators of the adverse effects caused by
such exposure, thus allowing knowing how much plants have
suffered from this type of environmental stress.
The recognition of the problem of HM pollution by international
authorities led to the implementation of stricter laws to control
discharges of these metals into the environment, and to the
development of technologies to solve this serious ecological
problem. Phytoremediation is an emerging technology that uses
plants and their rhizosphere-associated microbes for environmental cleanup [8e11]. Various phytoremediation strategies are
possible, with different phytotechnologies proting from different
plant properties. Concerning metal decontamination of soils, the
main treatment streamlines are phytostabilization, the use of
plants to stabilize pollutants in soil, either by preventing leaching
or erosion, or by converting pollutants into less bioavailable and
toxic [1,11], and phytoextraction, the use of plants to clean up
pollutants via extraction and accumulation of the toxic elements in
harvestable tissues [11e13]. A concept that has accompanied the
development of phytoextraction strategies is the so-called hyperaccumulators, which are plant species who are able to accumulate
in their harvestable tissues one or more inorganic elements, such as
HM, to levels 100-fold higher than other species [14].
Solanum nigrum L., commonly known as black nightshade, is
a plant species that has been reported to hyperaccumulate HM such as
cadmium and zinc, and has the particularity of being a fast growing,
easily adaptable plant and having a greater biomass than most
hyperaccumulators [15], making it a potential candidate for phytoremediation and for the accumulation of other metals, such as Ni.
Hyperaccumulation has been recognized as an extreme physiological response to heavy metal tolerance. Thus, to tolerate high
concentrations of metals in their tissues, plants use several metalbinding biomolecules, including low-molecular-weight ligands and
small metal-binding proteins, such as histidine, organic acids, phytosiderophores, phytochelatins and metallothioneins (MT), in order
to sequester, transport and store the accumulated metals [16e18].
MTs are low molecular weight, cysteine-rich, metal-binding
proteins, that are products of mRNA translation. The classication
of these proteins is one of the most controversial issues involving
its study [19]. Due to the high protein similarity between the

255

different MTs, the proposed classications are based on the

arrangement of their cysteine residues [20]. Although there are
several conicting classications, the most accepted and followed
by researchers is the classication of the various plant MTs into four
types: MT1 (subtypes a, b, c), whose gene expression is higher in
roots than shoots, MT2 (subtypes a, b, c, d), in which gene
expression occurs mostly in shoots, MT3 (subtypes a, b, c), that has
a specic accumulation of their transcripts in eshy fruits as they
ripen, and MT4, whose gene expression is restricted to developing
seeds [16,21]. Regarding S. nigrum MT system, the available
S. nigrum MT-encoding cDNA sequences at the NCBI database
(www.ncbi.nlm.nih.gov, 2010) belong only to MT types 2 and 3
(Fig. 1), which hinders the study of these proteins in this plant
species.
Although these proteins have been widely studied, their specic
contribution toward plant metal homeostasis is poorly known.
Nevertheless, there is some evidence of their involvement in
copper tolerance, and in the transport of cadmium and zinc [16],
which suggests that these proteins could be the peptide complexes
rst reported in 1988, which are involved in Ni transport in the
xylem [22]. Furthermore, recent studies have suggested that the
histidine residues of MT molecules may also be related to the
sequestration and transport of metal ions in plants [23].
Thus, it is possible that MTs have an important role in plant
homeostasis and detoxication of other HM, including Ni, and may
be involved in transporting and chelating such pollutants in plant
tissues, as previously described for the aquatic fern Azolla liculoides [24]. In this way, determining the real functions of these
proteins in higher plants remains a fascinating challenge.
This work focused on evaluating the Ni accumulation potential
of the Cd, Zn hyperaccumulator S. nigrum L., and the participation of
MTs in the plant Ni homeostasis. Several biometric and biochemical
parameters were determined in order to evaluate the effects of the
exposure to this metal in S. nigrum plants. The quantication of Ni
accumulation was also performed to verify if this plant can be
considered a hyperaccumulator of this HM and/or if it can be used
for phytoremediation purposes. RT-PCR semi-quantitative reactions were performed to investigate the specic accumulation of
MTs transcripts in roots and shoots, in order to discriminate the
participation of these proteins in S. nigrum Ni homeostasis, so that
their specic contribution toward HM homeostasis may be further
claried.
2. Results
2.1. Metal exposure effects on biometric parameters
At the end of the exposure period, Ni-treated plants showed
visible signs of metal toxicity, such as growth reduction. The
determined biometric parameters of Ni-exposed plants can be
observed in Fig. 2. Shoot fresh weight signicantly decreased by
52% with the 7.5 mM Ni treatment and 45% with the 100 mM Ni
shock treatment. Root fresh weight signicantly decreased by 65%
with both Ni treatments (Fig. 2A). Shoot dry weight signicantly
decreased by 61% and 54% in 7.5 mM and 100 mM Ni-treatments,

Fig. 1. Sequence alignments of the predicted protein sequence for the known S. nigrum MT-encoding cDNAs available at the NCBI database (2010): MT2 group and MT3 group.
Sequences were aligned by the ClustalW program [38]. Black boxes represent the cysteine domains used for MT classication according to Cobbett and Goldsbrough [16].

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P. Ferraz et al. / Plant Physiology and Biochemistry 57 (2012) 254e260

Fig. 3. Ni levels accumulated in shoots and roots of S. nigrum plants exposed to 7.5 mM
or 100 mM Ni. Columns represent mean S.D. of triplicates (n  3). Grey bars correspond to shoots (S) and white bars correspond to roots (R).

lipid peroxidation levels were higher compared to roots in all

treatments, and no signicant differences in MDA content in shoots
were detected among all situations. Root lipid peroxidation
signicantly increased only with the 100 mM Ni treatment, by
approximately 1.3 fold.
As shown in Fig. 4B, shoot free proline content from Ni-treated
plants signicantly increased only with the 7.5 mM Ni treatment, by
1.4 fold. In roots, the proline content signicantly increased only
with the 100 mM Ni treatment, by 1.3 fold.
No signicant differences were observed regarding carotenoids
and chlorophyll contents between all growth conditions used (data
not shown).

Fig. 2. Biometric parameters of control and Ni-treated S. nigrum plants. A) fresh

weight, B) dry weight, C) shoot and root length. Columns represent mean S.D. of
triplicates (n  3). S e shoots (grey bars); R e roots (white bars); C e control; 7.5 e
7.5 mM Ni; 100 e 100 mM Ni. Asterisks represent signicant differences at P < 0.05.

respectively. No signicant difference was detected in root dry

weight values (Fig. 2B).
Shoot length signicantly decreased by about 33% with the
7.5 mM Ni treatment and 30% with the shock treatment. Root length
decreased in both treatments, but only signicantly at 7.5 mM Ni
treatment, by 30% (Fig. 2C).
2.2. Metal accumulation
For each Ni exposure treatment (both prolonged and shock) it
was possible to verify a higher accumulation of Ni in roots than in
shoots. No signicant variations in Ni accumulation levels were
observed between both treatments with this metal (Fig. 3). No
accumulation of Ni was observed regarding control plants (data not
shown).
2.3. Biochemical determinations
Lipid peroxidation levels, determined by estimating the
malondialdehyde (MDA) content, can be observed in Fig. 4A. Shoot

Fig. 4. A) Lipid peroxidation levels, determined by estimating the MDA content, in

S. nigrum control plants and exposed to Ni. B) Proline content of S. nigrum control
plants and exposed to Ni. Columns represent mean S.D. of triplicates (n  3). S e
shoots (grey bars); R e roots (white bars); C e control; 7.5 e 7.5 mM Ni; 100 e 100 mM
Ni. Asterisks represent signicant differences at P < 0.05.

P. Ferraz et al. / Plant Physiology and Biochemistry 57 (2012) 254e260

2.4. MT mRNA accumulation analysis

Due to the high similarity between the available S. nigrum MTencoding cDNA sequences (types 2 and 3), in particular between
subtypes MT2a and MT2b, subtypes MT2c and MT2d, and between
all MT3 sequences, it was only possible to design specic sets of
primers for MT2b-, MT2c- and MT3c-encoding sequences. Since it
was not possible to obtain specic primers for the other known MT
sequences, another set of primers were designed to discriminate
the group MT2a b from the MT2c d and from the MT3 one.
Thus, comparing the results obtained with all these pairs of primers
it is possible to evaluate the accumulation of all types of S. nigrum
MT2- and MT3-encoding mRNAs. Regarding MT1, as there are no
available MT-encoding cDNA sequences, the strategy used was to
elaborate degenerated primers deduced from other MT1 sequences
available at the NCBI database. The limiting number of MT1encoding sequences available plus belonging to species that are
phylogenetically distant, added to the misclassication of some of
the existing ones while others were incomplete, have led to choose
the MT1-encoding cDNA sequences from Arabidopsis thaliana for
primer design.
The analysis by agarose gel electrophoresis of the semiquantitative RT-PCRs performed to evaluate the accumulation of
the different types of S. nigrum MT-encoding mRNAs can be
observed in Fig. 5A. The accumulation of MT1-related transcripts
occurred in both shoots and roots from plants of the control situation and these transcript levels remained constant in shoots from

257

both Ni treatments, and decreased in roots from both Ni treatments, predominantly in the 7.5 mM Ni treatment. No MT2b-related
transcripts were detected in both shoots and roots from all situations analyzed, but could be detected in seedlings (data not shown).
This analysis also showed an occurrence of a de novo accumulation
of MT2c-related transcripts only in shoots from both Ni treatments,
as none were detected in the control situation. No MT3c-related
transcripts were detected in both shoots and roots from all
analyzed situations. The accumulation of MT2a b- and MT2c drelated transcripts occurred in both shoots and roots from plants of
the control situation and these transcript levels increased with both
Ni treatments. The MT3-related transcripts accumulated only in
shoots of Ni-treated plants, predominantly in the 100 mM Ni
treatment.
3. Discussion
Exposure of S. nigrum to high levels of Ni induced various
deleterious effects on plant development with the appearance of
toxicity symptoms. Both Ni concentrations used in this study led to
a signicant decrease in root and shoot fresh weight, and in shoot
length and dry weight, with a signicant parallel increase in water
content (Fig. 2). Root length only decreased in the 7.5 mM Ni
treatment, as the roots of plants from the shock treatment were
already large enough at the onset of Ni exposure (the fourth week),
thus not being expected a greater growth of these organs
throughout the last week and therefore no signicant differences

Fig. 5. A) Analysis by a 1% (w/v) agarose gel electrophoresis of typical RT-PCR reactions performed for the detection of MT1-, MT2b-, MT2c-, MT3c-, MT2a b group-, MT2c d
group- and MT3 group-related transcripts in shoots (S) and roots (R) of S. nigrum control plants and exposed to Ni. B) Loading controls of the amount of total RNA used for the RTPCR procedures corresponding to 200 ng of RNA loaded and separated by 0.8% (w/v) agarose gel electrophoresis. C e control; 7.5 e 7.5 mM Ni; 100 e 100 mM Ni. Arrows indicate the
location and size of the corresponding expected amplied products.

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P. Ferraz et al. / Plant Physiology and Biochemistry 57 (2012) 254e260

were detected. These results show that high concentrations of Ni

cause growth inhibition of S. nigrums roots and shoots. Similar
results have already been described for the non-hyperaccumulators
rice (Oryza sativa L.) [5] and wheat (Triticum aestivum L.) [4]. This
inhibitory effect may be related to the competition of Ni in the
uptake of other essential metal ions, such as magnesium, iron,
copper and zinc [2,25], provoking their deciency and leading to
plant growth impairment.
Ni accumulation was always higher in roots than in shoots
(Fig. 3), evidencing a slow translocation rate of Ni between these
organs [26]. Because of this evident accumulation of Ni in S. nigrum
roots, this plant species cannot be considered a Ni hyperaccumulator because the quantied levels are lower than 1 mg g1
dry weight [27]. Nevertheless, it can be used in for Ni phytostabilization, preventing its leaching and further contamination of
natural watercourses.
Of the two Ni treatments, only the one-week 100 mM Ni exposure caused an increase in roots lipid peroxidation levels (Fig. 4),
indicating that these tissues have suffered oxidative membrane
damage. An increase in free proline levels in this exact situation was
also detected, which may contribute to protect root cells from the
damage caused by the Ni-induced oxidative stress [28]. Considering
the shoots of the 100 mM treatment, there were no signicant
differences in these two parameters, which combined with the
absence of changes in chlorophyll and carotenoids contents
suggests that oxidative stress did not occur in photosynthetic
tissues of 100 mM Ni-treated plants. However, there was an increase
in free proline content in shoots of plants treated with 7.5 mM Ni,
which can be related to the induction of its synthesis or an inhibition of its degradation in response to the metal, even though no
apparent oxidative stress was set, similarly to what has been
described for rice [28]. However, the participation of other antioxidant mechanisms, such as enzymes and other chelating agents,
must not be ruled out.
The MT mRNA accumulation analysis (Fig. 5) presented an
overview on the inuence of Ni in the regulation of MT1-, MT2- and
MT3-related transcript accumulation in shoots and roots from the
metal-exposed plants. MT1-related transcripts were constitutively
accumulated in plants from the control situation, and these transcript levels remained constant in shoots from both Ni treatments,
while a decrease of transcript levels occurs in the roots from Nitreated plants. This is a curious result, since the gene expression
of this type of MT was previously described as being higher in roots
than shoots [16]. This situation suggests that the other groups of
MTs may offset the decrease of MT1-related transcripts levels in
roots, the organ where the most of the Ni was accumulated.
MT2a- and MT2d-related transcripts were constitutively accumulated in plants from the control situation and the exposure to Ni
stimulated this accumulation in roots and shoots. Also, Ni induced
the accumulation of MT2c-related mRNAs only in shoots. These
results suggest that MT translocation may occur from aerial tissues
to roots. Because most of the Ni was accumulated in roots, and due
to the decrease of MT1-related transcripts levels in roots from the
Ni-treated plants; these translocated MT2 may play a protective
role against the harmful effects of Ni by chelating it and rendering
less harmful in these organs. Furthermore, these results suggest the
involvement of specic MT2 type subfamily members in the
defense process against the exposure to high Ni levels in both roots
and shoots.
MT2b-related transcripts could not be detected in any situation
analyzed, implying that the MT2b members do not participate in
metal homeostasis in full-grown plants.
Regarding MT3-related mRNA accumulation, an increased
transcript accumulation in shoots was observed only with the
exposure to Ni, especially in the shock-treated plants. Since no

MT3c-related transcripts were detected in both shoots and roots

from all analyzed situations, these results suggests that only MT3a
and/or MT3b were involved in the tolerance to high levels of Ni in
S. nigrum. Moreover to the Ni-induction, the accumulation of MT3related mRNAs in shoots was a surprising result, as these MTs were
described as being specic to the process of fruit ripening
[16,29e31].
According to the results obtained in this work, it is possible to
assume that Ni induced an increased accumulation of MT2a-,
MT2d-related transcripts in both roots and shoots, and the de novo
accumulation of the MT2c-, MT3-related transcripts only in shoots,
suggesting that these MTs are related to the Ni homeostasis in
S. nigrum, thus contributing to the clarication of the roles the
various types of MTs play in metal homeostasis in plants. Hence,
this study showed that S. nigrum can tolerate high concentrations of
Ni in the soil solution, uptake it and store it in their roots, and that
these plants used proline and specic MTs (and possibly other
protective agents) to protect themselves from the oxidative damage
caused by Ni exposure.
The present results also suggest that the actual classication of
plant MTs may not be the most appropriate, since the accumulation
of MT1-related mRNAs decrease in the roots of Ni-treated plants
and the accumulation of MT2-related transcripts were induced in
roots from Ni-treated plants. As previously mentioned, the accumulation of MT3-related mRNAs in shoots was an unexpected
result, as these MTs were described as being specic to the process
of fruit ripening. Taken together, these results demonstrate that this
classication is not linear and that the gene expression and the
transcript distribution in the plant body may vary depending on the
plant species. Furthermore, the fact that the histidine residues of
MT molecules may also play a role in metal transport in plants,
reinforces the idea that the classication based on the arrangement
of MT cysteine residues may not be sufciently adequate.
4. Methods
4.1. Growth conditions and biometric analysis
Fifteen days after germination, S. nigrum L. plants were grown
hydroponically in plastic pots, using a mixture of vermiculite and
perlite (2:1) as substrate under greenhouse conditions for four
weeks in a nutrient solution (Hoagland solution) [32] supplied
under 3 different situations: one set without Ni; another one
exposed to 7.5 mM Ni (as NiSO4$6H2O), simulating a soil contaminated with this metal; and the third consisted on a short shock
treatment with 100 mM Ni throughout the last week, simulating
a discharge of high concentrations of this pollutant to an uncontaminated soil. These metal concentrations were set in preliminary
seed germination tests in the nutrient media supplemented with
increasing concentrations of Ni (data not shown).
At the end of the treatment period, at least 3 plants from each
condition were used for the determination of several biometric
parameters: root and shoot fresh weight, dry weight and length.
Roots and shoots from these plants were frozen and grinded with
liquid N2, and the resulting powder was stored at 80  C until used.
4.2. Metal accumulation analysis
Root and shoot samples were washed with tap water, followed
by further washing with HCl 0.1 M and demineralized water. Then,
the plant material was oven-dried (60  C), grinded to a ne powder
and stored at room temperature until used. For Ni determination,
a microwave-assisted acid digestion in closed vessels was performed, originating acid extracts that were analyzed by inductively
coupled plasmaemass spectrometry (ICPeMS). Briey, sample

P. Ferraz et al. / Plant Physiology and Biochemistry 57 (2012) 254e260

masses up to 500 mg were placed in microwave oven vessels and

3 mL of concentrated (65% w/v) HNO3 (Suprapur, Merck) and
1.5 mL of 30% (w/v) H2O2 (Fluka) were added. Then, the vessels
were sealed and heated in the microwave unit (a Milestone MLS
1200 mega, equipped with an HPR-1000/10 S rotor) according to
the following time (min.)/power (W) program: 1/250 e 2/0 e 5/250
e 5/400 e 5/600. After cooling, the vessels content were transferred into 25 mL volumetric asks and diluted to volume with
ultrapure water (Milli-Q, Millipore). ICPeMS analysis was performed using a VG Elemental, PlasmaQuad 3 (quadrupole-based)
instrument, equipped with concentric glass nebulizer (Meinhard
Type A), water-cooled glass spray chamber with impact-bead,
standard quartz torch and nickel skimmer and sampling cones.
For sample introduction, a Minipuls 3 (Gilson) peristaltic pump was
used. 60Ni was monitored as analytical isotopes and 45Sc and 89Y
as internal standards. Results were expressed as mg of metal g1 of
sample (dry weight).

259

The forward primer and reverse primer sequences, melting

temperature (TM) and the expected amplied size from each group
of MTs analyzed are:
MT1: 50 e GCA GTT GCG AGA AGA ACT AC e 30 , 50 e CCA GTG AGC
AGA GTG ACG AGG ACT CGA GCT CAA GC e 30 , TM 52  C, z380 bp;
MT2b: 50 e GGG ATC CGA TTA TGT CTT G e 30 , 50 e ATT ACC AGA AGC
AGA GAT GC e 30 , TM 46  C, 433 bp; MT2c: 50 e GAT GTG GGA TGT
ACC CTG AC e 30 , 50 e GTT ACA AGC CCA TGT CAA CTT C e 30 ,
TM 49  C, 362 bp; MT3c: 50 e GTC GGA CAA GTG TAG TAG TTG e 30 ,
50 eAGA CCA AAG AGA CAG ACT AGA G e 30 , TM 44  C, 340 bp;
MT2ab: 50 e GCT GTG GAG GAT GCA AGA T e 30 , 50 e CTT AGA GCA
AGT GCA AGG GTT AC e 30 , TM 50  C, 191 bp; MT2cd: 50 e GAT
GTG GGA TGT ACC CTG AC e 30 , 50 e GCA GTT TGA TCC ACA TTT
GC e 30 , TM 49  C, 146 bp; MT3: 50 e TGC TGA TGT CAG CCA
ATG e 30 , 50 e CCA GTG AGC AGA GTG ACG AGG ACT CGA GCT CAA
GC e 30 , TM 48  C, 440 bp.
As MT4 are exclusive from seeds, this group was not considered
in this study.

4.3. Lipid peroxidation

Lipid peroxidation levels in roots and shoots were determined
by estimating the malondialdehyde (MDA) content as described by
Ref. [33]. About 400 mg of stored frozen powder were used for each
reaction. The concentration of MDA was calculated using the
extinction coefcient of 155 mM1 cm1 and results were
expressed as nmol MDA g1 fresh weight.
4.4. Proline content
Proline was quantied as described by Bates et al. [34]. Proline
was extracted from plant samples by homogenization of
150e200 mg of frozen powder in 3% (w/v) sulphosalicylic acid with
quartz sand. Results were expressed as mg proline g1 fresh weight.
4.5. Chlorophylls and carotenoids content
Photosynthetic pigments from 120 to 150 g of shoots frozen
samples were extracted in 80% (v/v) acetone with quartz sand. The
homogenate was centrifuged at 2150 g for 10 min. After centrifugation, the absorbance of the supernatant was measured at 470,
647 and 663 nm, and chlorophylls and carotenoids contents were
estimated from the formulas of Lichtenthaler [35]. Results were
expressed as mg g1 fresh weight.
4.6. Primers design
Available S. nigrum MT-encoding cDNA sequences (www.ncbi.
nlm.nih.gov, 2010): MT2a (GenBank ID: EU760481.1); MT2b (GenBank ID: EU760482.1); MT2c (GenBank ID: EU760483.1); MT2d
(GenBank ID: EU760484.1); MT3a (GenBank ID: FJ546423.1); MT3b
(GenBank ID: FJ546424.1); MT3c (GenBank ID: FJ546425.1), were
aligned (Blastn), and the resulting alignments were submitted to
the PrimerIdent software (http://primerident.up.pt, 2010) [36] for
MT-specic primers design. Due to the high similarity between the
available S. nigrum MT sequences, it was only possible to design
specic sets of primers (forward and reverse) for MT2b-, MT2c- and
MT3c-encoding sequences. Another set of primers were designed
to discriminate the group MT2a b from the MT2c d and from
the MT3 one, as previously described in section 2.4. Regarding MT1,
as there are no available S. nigrum MT-encoding cDNA sequences at
the NCBI database, the strategy used was to elaborate degenerated
primers deduced from MT1-encoding cDNA sequences from A.
thaliana: MT1a (GenBank ID: NM_100633.2); MT1b (GenBank ID:
NM_001037008.2); MT1c (GenBank ID: NM_100634.1).

4.7. RNA extraction and MT RT-PCRs

Total RNA extraction was performed using the Trizol reagent
(Invitrogen, USA), according to the manufacturer instructions. RNA
concentration and quality were spectrophotometrically assessed
and electrophoretically conrmed, respectively, and RNA preparations were stored at 80  C until used.
Analysis of MT transcript accumulation was performed by
optimized semi-quantitative RT-PCRs [37]. RT reactions for each
treatment/organ were performed using the Reverase (M-MuL V RT)
reagents (Bioron, Germany), according to the instructions supplied.
The RT procedure used 5 mg of total RNA as starting template, and
1 mg of the primer complementary to the poly-A tail 50 e TTT TTT
TTT TTT TCG AAC TCG AGC TCA GGA GCA GTG AGA CGA GTG
ACC e 30 . RT nal reactions were kept at 80  C until needed.
PCR was performed in a 25 mL reaction composed of 1 mL of the
RT reaction, 1x PCR complete buffer (with MgCl2), 0.2 mM dNTPs,
0.1 mM of forward primer, 0.1 mM of reverse primer and 1.25 U of Taq
DNA Polymerase (Fermentas, Lithuania). The PCR reactions were
set on a Mastercycler Gradient thermocycler (Eppendorf, USA) with
the following optimized program: an initial denaturation step at
94  C for 1 min, followed by 35 cycles of 94  C e 3000 , TM (specic
for each primer pairing) e 3000 , 72  C e 3000 . The nal extension, for
completing uncompleted DNA strands, was performed at 72  C for
3 min. At least 3 RT-PCR reactions were performed for each
analyzed mRNA. Reaction mixtures were analyzed by 1% (w/v)
agarose gel electrophoresis. All gels were captured using the
Kodak EDAS 290 imaging system and the Kodak 1D software
v.3.5.4 (Kodak, USA).

4.8. Statistical analysis

Biometric parameters, biochemical determinations, and metal
accumulation assays were performed at least in triplicate (n  3).
Variance analysis was performed by Fisher test and the means were
statistically analyzed using a two-sided t-test. Statistical signicance is assumed at P  0.05.

Acknowledgments
The authors gratefully acknowledge the University of Porto for
nancial support (Project MetalloChromium, IJUP 2010/11) with
the contribution of Santander Totta.

260

P. Ferraz et al. / Plant Physiology and Biochemistry 57 (2012) 254e260

References
[1] S. Eapen, S.F. DSouza, Prospects of genetic engineering of plants for phytoremediation of toxic metals, Biotechnol. Adv. 25 (2005) 97e114.
[2] C. Chen, D. Huang, J. Liu, Functions and toxicity of nickel in plants: recent
advances and future prospects, Clean 37 (2009) 304e313.
[3] F.W. Sunderman, A. Oskarsson, Nickel, in: E. Merian (Ed.), Metals and Their
Compounds in the Environment: Occurrence, Analysis and Biological Relevance, VCH, Weinteim, Cambridge, 1991, pp. 1101e1126.
[4] E. Gajewska, M. Sklodowska, M. Slaba, J. Mazur, Effect of nickel on antioxidative enzyme activities, proline and chlorophyll contents in wheat shoots,
Biol. Plant 50 (2006) 653e659.
[5] Y.C. Lin, C.H. Kao, Nickel toxicity of rice seedlings: cell wall peroxidase, lignin,
and NiSO4-inhibited root growth, crop, Environ. Bioinform. 2 (2005) 131e136.
[6] S.A.L. Andrade, P.L. Grato, M.A. Schiavinato, A.P.D. Silveira, A.A. Azevedo,
P. Manzzafera, Zn uptake, physiological response and stress attenuation in
mycorrhizal jack bean growing in soil with increasing Zn concentrations,
Chemosphere 75 (2009) 1363e1370.
[7] S.S. Gill, N. Tuteja, Reactive oxygen species and antioxidant machinery in
abiotic stress tolerance in crop plants, Plant Physiol. Biochem. 48 (2010)
909e930.
[8] M.M. Lasat, Phytoextraction of toxic metals: a review of biological mechanisms, J. Environ. Qual. 31 (2002) 109e120.
[9] D.E. Salt, M.J. Blaylock, N.P.B.A. Kumar, V. Dushenkov, B.D. Ensley, I. Chet,
I. Raskin, Phytoremediation: a novel strategy for the removal of toxic metals
from the environment using plants, Nat. Biotechnol. 13 (1995) 468e474.
[10] D.E. Salt, R.D. Smith, I. Raskin, Phytoremediation, Annu. Rev. Plant Physiol.
Plant Mol. Biol. 49 (1998) 643e668.
[11] E. Pilon-Smits, Phytorremediation, Annu. Rev. Plant Biol. 56 (2005) 15e39.
[12] M.J. Blaylock, J.W. Huang, Phytoextraction of metals, in: I. Raskin, B. Ensley
(Eds.), Phytoremediation of Toxic Metals. Using Plants to Clean up the Environment, Wiley, New York, 2000, pp. 53e70.
[13] G.S. Bauelos, D.W. Meek, Accumulation of selenium in plants grown on
selenium-treated soil, J. Environ. Qual. 19 (1990) 772e777.
[14] R.D. Reeves, Hyperaccumulation of nickel by serpentine plants, in: J. Proctor,
A. Baker, R. Reeves (Eds.), The Vegetation of Ultramac (Serpentine) Soils,
Intercept Ltd., Andover, 1992, pp. 253e277.
[15] A.P.G.C. Marques, R.S. Oliveira, A.O.S.S. Rangel, P.M.L. Castro, Zinc accumulation in Solanum nigrum is enhanced by different arbuscular mycorrhizal
fungi, Chemosphere 65 (2006) 1256e1263.
[16] C. Cobbett, P.B. Goldsbrough, Phytochelatins and metallothioneins: roles in
heavy metal detoxication and homeostasis, Annu. Rev. Plant Biol. 53 (2002)
159e182.
[17] D.L. Callahan, A.J.M. Baker, S.D. Kolev, A.G. Wedd, Metal ion ligands in
hyperaccumulating plants, J. Biol. Inorg. Chem. 11 (2006) 2e12.
[18] L. Kerkeb, U. Krmer, The role of free histidine in xylem loading of nickel in
Alyssum lesbiacum and Brassica juncea, Plant Physiol. 131 (2003) 716e724.

[19] E. Freisinger, Plant MTs e long neglected members of the metallothionein

superfamily, Dalton Trans. 47 (2008) 6663e6675.
[20] G.M. Cherian, H.M. Chan, Biological functions of metallothioneins: a review,
in: K. Suzuki, N. Imura, M. Kimura (Eds.), Metallothionein III: Biological Roles
and Medical Implications, Birkhauser Verlag, Basel, 1993, pp. 87e109.
[21] N.J. Robinson, A.M. Tommey, C. Kuske, P.J. Jackson, Plant metallothioneins,
Biochem. J. 295 (1993) 1e10.
[22] D.A. Cataldo, K.M. McFadden, T.R. Garland, R.E. Wildung, Organic constituents
and complexation of nickel(II), iron(III), cadmium(II), and plutonium(IV) in
soybean xylem exudates, Plant Physiol. 86 (1988) 734e739.
[23] A. Torreggiani, A. Tinti, Raman spectroscopy a promising technique for
investigations of metallothioneins, Metallomics 2 (2010) 246e260.
[24] T. Schor-Fumbarov, P.B. Goldsbrough, Z. Adam, E. Tel-Or, Characterization and
expression of a metallothionein gene in the aquatic fern Azolla liculoides
under heavy metal stress, Planta 223 (2005) 69e76.
[25] B.Y. Khalid, J. Tinsley, Some effects of nickel toxicity on rye grass, Plant Soil 55
(1980) 139e144.
[26] S.K. Panda, S. Choudhury, Chromium stress in plants, Braz. J. Plant Physiol. 17
(2005) 95e102.
[27] U. Krmer, Metal hyperaccumulation in plants, Annu. Rev. Plant Biol. 61
(2010) 517e534.
[28] Y.C. Lin, C.H. Kao, Proline accumulation induced by excess nickel in detached
rice leaves, Biol. Plant 51 (2007) 351e354.
[29] S.K. Clendennen, G.D. May, Differential gene expression in ripening banana
fruit, Plant Physiol. 115 (1997) 463e469.
[30] S.E. Ledger, R.C. Gardner, Cloning and characterization of ve cDNAs for genes
differentially expressed during fruit development of kiwifruit (Actinidia deliciosa var. deliciosa), Plant Mol. Biol. 25 (1994) 877e886.
[31] S.J. Reid, G.S. Ross, Up-regulation of two cDNA clones encoding metallothioneinlike proteins in apple fruit during cool storage, Physiol. Plant 100 (1997) 183e189.
[32] D.R. Hoagland, D.I. Arnon, The Water-culture Method for Growing Plants
Without Soil. Circular 347, Univ. of Calif. Agric. Exp. Station, Berkley, 1950.
[33] R.L. Heath, L. Packer, Photoperoxidation in isolated chloroplasts, Arch. Biochem. Biophys. 125 (1968) 189e198.
[34] L.S. Bates, R.P. Waldren, I.D. Teare, Rapid determination of free proline for
water-stress studies, Plant Soil 39 (1973) 205e207.
[35] H.K. Lichtenthaler, Chlorophylls and carotenoids: pigments of photosynthetic
biomembranes, Methods Enzymol. 148 (1987) 350e382.
[36] A.M. Pessoa, S. Pereira, J. Teixeira, PrimerIdent: a web based tool for conserved
primer design, Bioinformation 5 (2010) 52e54.
[37] J. Teixeira, F. Fidalgo, Salt stress affects glutamine synthetase activity and
mRNA accumulation on potato plants in an organ-dependent manner, Plant
Physiol. Biochem. 47 (2009) 807e813.
[38] M.A. Larkin, G. Blackshields, N.P. Brown, R. Chenna, P.A. McGettigan,
H. McWilliam, F. Valentin, I.M. Wallace, A. Wilm, R. Lopez, J.D. Thompson,
T.J. Gibson, D.G. Higgins, Clustal W and Clustal X version 2.0, Bioinformatics 23
(2007) 2947e2948.