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1. A plant genetically modified that accumulates Pb is especially
promising for phytoremediation
Carmina Gisbert,a
Roc Ros,b
Antonio De Haro,c
David J. Walker,d
M. Pilar Bernal,d
Ram
o
on Serrano,a
and Juan Navarro-Avi~
n
n
o
oa,*
a
Departamento de Biolog
ı
ıa del estr
e
es, IBMCP, CSIC, Camino de Vera s.n., Valencia 46022, Spain
b
Departamento de Fisiolog
ı
ıa Vegetal, Universidad de Valencia, Campus de Burjasot, Burjasot, Valencia 46100, Spain
c
Departamento de Agronom
ı
ıa y Mejora Gen
e
etica Vegetal, Instituto de Agricultura Sostenible, CSIC, Alameda del Obispo s.n., C
o
ordoba 14080, Spain
d
Departamento de Conservaci
o
on de Tierra y Agua y Administraci
o
on de Residuos Org
a
anicos, Centro de Edafolog
ı
ıa y Biolog
ı
ıa Aplicada del Segura,
CSIC, Campus de Espinardo, Apartado 4195, Murcia 30080, Spain
Received 21 February 2003
Abstract
From a number of wild plant species growing on soils highly contaminated by heavy metals in Eastern Spain, Nicotiana glauca R.
Graham (shrub tobacco) was selected for biotechnological modification, because it showed the most appropriate properties for
phytoremediation. This plant has a wide geographic distribution, is fast-growing with a high biomass, and is repulsive to herbivores.
Following Agrobacterium mediated transformation, the induction and overexpression of a wheat gene encoding phytochelatin
synthase (TaPCS1) in this particular plant greatly increased its tolerance to metals such as Pb and Cd, developing seedling roots
160% longer than wild type plants. In addition, seedlings of transformed plants grown in mining soils containing high levels of Pb
(1572 ppm) accumulated double concentration of this heavy metal than wild type. These results indicate that the transformed
N. glauca represents a highly promising new tool for use in phytoremediation efforts.
Ó 2003 Elsevier Science (USA). All rights reserved.
Keywords: Cadmium; Hyperaccumulators; Lead; Nicotiana glauca; Phytochelatin synthase; Bioremediation
Toxic metal pollution of the biosphere has acceler-
ated rapidly since the onset of the industrial revolution
and heavy metal toxicity poses major environmental and
health problems. It is estimated that cleanup of haz-
ardous wastes using conventional technologies will cost
at least $200 billion in the US alone [1]. Lead is one of
the most frequently encountered heavy metals in pol-
luted environments. The primary sources of this metal
include mining and smelting of metalliferous ores,
burning of leaded gasoline, disposal of municipal sew-
age, and industrial wastes enriched in Pb as well as using
of Pb-based paint [2]. The threat that heavy metals pose
to human and animal health is aggravated by their long-
term persistence in the environment. For instance, Pb
one of the more persistent metals, was estimated to have
a soil retention time of 150–5000 years [3]. Also, the
average biological half-life of cadmium has been esti-
mated to be about 18 years [4].
The use of biological materials to cleanup heavy
metal contaminated soils has been focused on as an ef-
ficient and affordable form of bioremediation. Phyto-
remediation is an emerging and low cost technology that
utilizes plants to remove, transform, or stabilize con-
taminants located in water, sediments, or soils. Vegeta-
tion growing in contaminated sites has developed a
mechanism of tolerance to the inadequate environments,
therefore a well adapted flora, tolerant to edafoclimatic
conditions can be a basic instrument for phytoremedi-
ation [5,6]. Different biochemical studies of heavy metal
transport in plants have been conducted [7]. Plants
termed as phytoremediators are capable of absorbing
large amounts of heavy metals from the soil and accu-
mulating these metals in plant tissues [8,9]. One type of
metal-tolerant plant is the hyperaccumulator. This kind
of plant is known to be capable of both growing on soils
Biochemical and Biophysical Research Communications 303 (2003) 440–445
www.elsevier.com/locate/ybbrc
BBRC
*
Corresponding author. Fax: +34-96-387-78-59.
E-mail address: jpavinyo@upvnet.upv.es (J. Navarro-Avi~
n
n
o
o).
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S0006-291X(03)00349-8

2. contaminated with toxic metals and accumulating ex-
traordinary high levels of them (more than 0.1% of
heavy metal by dry weight in plant tissue, 0.01% for
cadmium). Up to date over 400 different hyperaccumu-
lator species have been identified [5].
No one knows why some plants accumulate metals
instead of keeping them out. One theory is that metals
keep insect pests at bay by deterring them from feeding.
Whatever the reason may be, hyperaccumulators could
result to be very useful in cleanup operations, because
they take metals out of the soil and store them in parts
of the plant above the ground. Metal-rich shoots and
leaves could simply be harvested and disposed off, in
landfill sites (for example). It might even be possible to
extract and recycle the metals [10]. The problem of all
these hyperaccumulators is that they have small biomass
and are slow-growing, therefore it could take many
years to decontaminate a polluted place. The ideal
phytoextractor should: (i) grow rapidly, (ii) produce
high amount of biomass, (iii) tolerate and accumulate
high concentrations of toxic metals, and (iv) contain
substances that deter herbivores from feeding, thus
preventing the heavy metal transfer to the food chain.
One possible solution might consist in expressing metal-
accumulating genes in nonaccumulating plants showing
interesting skills for bioremediation in order to turn
them into hyperaccumulator plants.
The aim of this work was to carry out the first step in
engineering metal tolerance in a selected wild plant
species, Nicotiana glauca R. Graham, which is fast-
growing, of high biomass, and tolerant of a wide range
of environmental conditions. This involved transfor-
mation of the plants with the wheat PC synthase gene
(TaPCS1), and subsequent determination of their heavy
metal accumulation and tolerance, specifically Pb and
Cd.
Materials and methods
Agrobacterium strain, plants transformation, and culture conditions
The binary Ti vector pBI121 (Clontech) was used for transforma-
tion. The GUS gene of the binary vector was replaced with the wheat
phytochelatin synthase 1 cDNA (TaPCS1, Accession No. AF093752)
to gain the new construct pBITaPCS1. The TaPCS1 cDNA (donation
of Professor Julian Schroeder, University of California, San Diego)
was originally cloned in pYES2 (Invitrogen) and designated pY-
ESTaPCS1. The plasmid was digested with XhoI and converted to
blunt ends with the DNA polymerase I (Klenow fragment). After-
wards, pYESTaPCS1 was digested with BamHI to produce a 2-kb
insert containing the TaPCS1 cDNA. PBI121 was digested with
BamHI and ECL136II. The 2-kb TaPCS1 insert was ligated to the
BamHI–Ecl136II sites of plasmid pBI121. The new construct (pBI-
TaPCS1) was electroporated into Agrobacterium tumefaciens strain
C58C1RifR
Rif [11].
Nicotiana glauca leaf explants were infected with A. tumefaciens
after two days of preculture on organogenic medium NB2510 [MS
salts [12] including Gamborg B5 vitamins (DUCHEFA), 3% sucrose,
2:5lgmL1
naphthalene acetic acid (NAA), 1lgmL1
of 6 benzyl
aminopurine (BA), and 0.8% agar (bacteriologic agar ‘‘Europeo’’
PRONADISA) in the dark. Explants from adult and young leaves
were infected by immersion on Agrobacterium culture for 10 min.
After 1 day of cocultivation, explants were transferred to selection
medium NB2510 containing 100lgmL1
kanamycin and 350lgmL1
carbenicillin. Two months after infection, shoots were individually
removed from the call using explants and transferred to bottles con-
taining 30 ml of B1 medium (MS salts including Gamborg B5 vita-
mins, 0:3lgmL1
indol acetic acid or 0:2lgmL1
NAA, 1% sucrose,
100lgmL1
, and 0.7% plant agar). Cultures were incubated in a
growth chamber (24–26 °C, 16 h light at 120lmolm2
s1
photon flux
density; Grolux, Sylvania, fluorescent tubes). Regenerated plantlets
were acclimatized in pots (25 cm diameter) with a mixture of peat and
vermiculite (3:1) in a growth incubator (25–27 °C, 16 h light at
71lmolm2
s1
photon flux density, and 62% relative humidity) and
then transferred to the greenhouse. Progenies were obtained from
those transgenic plants by selfing in controlled conditions and were
further characterized by genomic PCR, Southern and Northern blot
analyses (data not shown).
Plant growth and tolerance experiments
Experiment I (seedlings on agar medium). T2 seeds from transgenic
lines 1, 2, and 3 and wild type N. glauca were sterilized by rinsing in
40% sodium hypochlorite (50 g of active chlorine) for 10 min and
subsequently in sterile deionized water for 10 and 15 min. Fifty ster-
ilized seeds were sown in squared plates on medium PM (1.69 mM
CaðNO3Þ2H2O; 16.8 mM KNO3; 10gL1
sucrose; 7gL1
agar; pH 5).
After sterilization by filtration of a solution of CdCl2 and PbðNO3Þ2 we
obtained plates with 0, 0.15, 0.20, or 0.25 mM for CdCl2 and 0, 0.4, 0.8,
and 1.2 mM for PbðNO3Þ2. Medium pH was adjusted to 5. After 9 days
at 25 °C, 16 h light at 120lmolm2
s1
photon flux density (Grolux,
Sylvania, fluorescent tubes) the individual seedlings were harvested and
the length of roots was measured.
Experiment II (mature plants in contaminated soil). T2 seeds of
transgenic lines 1, 2, and 3 and wild type were germinated in petri
dishes with a medium prepared with 6gL1
agar, MS salts [12], and
10gL1
sucrose at pH 5.7 buffered with 0:25gL1
MES (2-[N-mor-
pholino]ethanesulfonic acid). Ten days after (when the first leaves were
developed) plantlets were transplanted to soil in pots (250 g soil) with a
metal contaminated soil (M4) diluted 50% (v/v) with vermiculite and a
control soil (peat pH adjusted to 6.0 with dolomite and vermiculite
50% v/v). Plants were grown in a culture growth chamber (25 °C, 17 °C,
day and night 16 h light). A minimum of 4 plants per soil were grown, 6
weeks old seedlings of transgenic, and wild type lines were divided into
roots and shoots, and fresh weight was determined. After elimination
of the soil, roots were washed with water, then with CaCl2–HCl (pH
3.8) for 10 min, and then with deionized water. Shoots were washed
with deionized water. Both shoots and roots were dried and analyzed
for heavy metals. Metal analysis was carried out by atomic absorption
spectrometry (AAS) after washing plant material at 480 °C for 6 h, and
then dissolved in HNO3 0.6 M.
Since soils from Valencia were contaminated with a high number of
heavy metals, the contaminated soil ‘‘M4’’ was obtained from close to
an old Pb–Zn mine at La Union, in the province of Murcia (SE Spain)
[13]. The M4 soil is a calcareous sandy-loam with 19.2% clay, 14.4%
silt, 66.4% sand, and a water holding capacity (WHC) of 144gkg1
.
The soil is a Xeric Torriorthent [15]. It has total concentrations of Pb
and Zn (1572 and 2602mgkg1
, respectively) which exceed greatly the
European Union maximum permitted levels for agricultural soils [14].
The soil was collected from the top 20 cm, air-dried for 5–6 days, and
sieved to 4 mm for pot experiments and to 2 mm for analysis. Total
heavy metals were extracted by nitric acid–perchloric acid digestion
[16]. All metal concentrations, adjusted to values for oven-dried (12 h
at 105 °C) soil, were determined by AAS. Full details of the analytical
procedures are given in [13].
C. Gisbert et al. / Biochemical and Biophysical Research Communications 303 (2003) 440–445 441

3. Results
Selection of a natural plant species specially good for
phytoremediation
Soils highly contaminated by heavy metals over sev-
eral decades were studied, searching for plant species
particularly useful for phytoremediation of this area and
others with similar agro climatic characteristics (manu-
script submitted). Heavy metal concentrations were de-
termined for soils and plant tissues collected from
contaminated sites. Most of the analyzed soils were lo-
cated in the metropolitan area of Valencia city (manu-
script submitted). Pb concentrations in these soils
ranged from 31 to 25,000 ppm. Cd ranged from 2 to
63 ppm. Other metals like Cr, Co, Ni, Cu, Zn, Hg, and
As were present at extremely high concentrations. For
instance, As was present at more than 12,000 ppm, Cr
up to 400 ppm, and Zn at more than 10,000 ppm. Some
of the most promising species were selected for further
studies and classified following a set of priorities: low
nutrient requirements, heavy metal tolerance, high bio-
mass, low water requirements, ease of vegetative prop-
agation, powerful root system capable of absorbing
metals from beneath the soil surface layers, suitability
for monoculture, and repulsion of herbivores. Among
the studied species, N. glauca (shrub tobacco) fulfilled all
the requirements and was selected for further studies.
Interestingly this plant species spans extended geo-
graphic areas such as Europe, Australia, and continental
America, which favors its possible application for bio-
remediation in many countries, increasing therefore its
commercial value. Besides, all the individuals of N.
glauca used in this study were isolated originally from a
contaminated site of this metropolitan area of Valencia
city, therefore it is likely that they are tolerant of ele-
vated metal concentrations.
Insertion of TaPCS1 gene increases lead uptake and
accumulation
The next step was to achieve the Agrobacterium
mediated DNA transmission (for direct gene shift) to N.
glauca. Transforming was pursued and finally achieved
(see Materials and methods) by incorporating and
overexpressing the gene TaPCS1, which encodes a
wheat phytochelatin synthase, shown to increase cad-
mium tolerance in yeast [17]. Phytochelatins have the
structure (l-Glu–Cys)n-Gly, where n has been reported
as being as high as 11, but is generally in the range 2–5
[18,19]. This enzyme is synthesized from glutathione,
having the ability to chelate heavy metals [20]. Once the
metal is chelated, it is transported to the vacuole where
it is properly disposed. After transformation, seedling
growth was tested in both wild type and modified lines
(see Materials and methods), to investigate whether the
Pb tolerance conferred by TaPCS1 may be found also in
plant tissues. In Fig. 1A, it can be seen that genetically
modified plants (TaPCS1 insertion was confirmed by
genomic PCR, Southern, and Northern blot analyses;
data not shown) showed higher Pb tolerance. Roots
growth was improved drastically (near 160%) and leaves
were higher and greener in the transformed plants in the
presence of 0.8 mM lead. The increased tolerance to lead
was observed in a range of lead concentrations up to
1 mM (Fig. 1B). For all the lines containing TaPCS1
gene (the 3 plant lines used in this study were from
separate transformation events), and in almost all the
Fig. 1. Seedling growth in control and high-Pb media. Left panel: wild type (w), and modified lines (1 and 2), grown for 10 days in agar media
containing no added Pb (upper line) or 0.8 mM PbðNO3Þ2 (lower line). Root lengths (right panel) are displayed in mm (w: wild type, 1, 2, and 3 are
lines including TaPCS1). Twenty-five seedlings were measured for each line. For lines 1, 2, and 3, the segregation ratio was 16:1. v2
values: 0.78, 0.09,
and 0.19, respectively.
442 C. Gisbert et al. / Biochemical and Biophysical Research Communications 303 (2003) 440–445

4. concentrations tested roots were longer than those of
wild type plants, even when they were shorter in the Pb
medium (line 3), displaying maximum differences at
0.8 mM PbðNO3Þ2. However, whether this increase in
tolerance is due to greater metal exclusion or higher lead
absorption cannot be inferred from the obtained results.
For this reason wild and modified plants were grown
during 6 weeks in a metal contaminated soil taken from
a mining area of Murcia, Spain (Table 1) and the Pb
concentrations in plant tissues were determined. Lead
concentration was increased by an average of about 50%
(51:21 5:6; 52 ppm in ashes) in the aerial part of
genetically modified plants (Fig. 2). Besides, lead ab-
sorption reaches up by about a 85% (82:68 4) incre-
ment in the roots of plants containing the gene TaPCS1
(981 ppm in ashes). These results confirm the initial
presumption that overexpression of TaPCS1 might in-
crease lead tolerance and absorption in N. glauca, the
plant species chosen for bioremediation purposes.
Genetically modified plants are more tolerant to Cd
When gene TaPCS1 was expressed in a wild type strain
of Saccharomyces cerevisiae [17] it conferred increased
tolerance to Cd. This is true for N. glauca plants as well.
Fig. 3 illustrates the Cd toxicity effect on plant roots de-
veloped supplying 50lM CdCl2 to N. glauca. Seedlings
overexpressing the gene TaPCS1 had longer roots (near
160%) and higher and greener leaves than unmodified
plants, resembling the behavior of seedlings supple-
mented with PbðNO3Þ2. As expected CdCl2 incites higher
toxicity than PbðNO3Þ2 for the reason that the concen-
tration range used to test out the seed-tolerance to Cd,
comes near three orders of magnitude less. The greatest
differences in growth between wild type and transformed
Table 1
Characteristics of the soil (M4) used in pot experiments
Characteristic M4 EUa
pH 7.70 6–7
EC (dS m1
) 2.21 —
CaCO3 (%) 28.0 —
CEC (cmolc kg1
) 10.6 —
OM (%) 0.65 —
Organic-C (gkg1
) 3.8 —
Total-N (gkg1
) 0.3 —
Fe (gkg1
) 53.1 —
Mn (gkg1
) 1.6 —
Cu (mgkg1
)a
42 50–140
Pb (mgkg1
)a
1572 50–300
Zn (mgkg1
)a
2602 150–300
Ni (mgkg1
)a
2.0 30–75
Cd (mgkg1
)a
0.2 1–3
Cr (mgkg1
)a
2.0 100–150
a
European Union limits (mgkg1
) for agricultural soils [14].
Fig. 2. Percent of increment in internal Pb concentration. Seedlings of
wild type (w) and modified (m) plants growing in mine soil containing
1572mgkg1
Pb, were analyzed after separation of the aerial parts and
the roots. Results are the average of three different experiments.
Standard deviation is less than 10%.
Fig. 3. Seedling growth in control and high-Cd media. Left panel: wild type (w) and modified lines (3 and 2), grown for 9 days in agar media
containing no added Cd (upper line) or 50 mM CdCl2 (lower line). Root lengths (right panel) are displayed in mm (w: wild type, 1, 2, and 3 are lines
including TaPCS1).
C. Gisbert et al. / Biochemical and Biophysical Research Communications 303 (2003) 440–445 443

5. lines were obtained at a 30 lM Cd (Fig. 3). This confirms
the hypothesis that increased TaPCS1 expression could
mediate also a higher Cd tolerance in N. glauca.
Discussion
Phytoremediation, the use of plant material to clean
up heavy metal contaminated soils, has been focused on
as an efficient and affordable form of bioremediation
[21,22]. The conventional technologies used for in situ
and ex situ remediation are typically expensive and de-
structive [23]. Hyperaccumulators such as Thlaspi and
Alyssum species [5] emerge, at first, as an obvious way to
approach the question, however, this type of plant spe-
cies have important limitations. First, hyperaccumula-
tors are usually specific for one particular metal [5], and
are adapted to precise climate and soil conditions (not
really transferable). Furthermore, they cannot be man-
aged as a conventional crop, have low biomass, and often
a short life cycle. Therefore it seems more reasonable to
search for nonhyperaccumulator plants showing good
features for phytoremediation and then transfer bio-
technologically traits that make the modified plant even a
more powerful tool than natural hyperaccumulators.
Hence, the aim of the work reported in this paper was to
engineer increased heavy metal absorption in a screen-
selected wild type plant species. The studies have focused
on two of the most worrisome contaminants: Pb and Cd.
Up to now, different plant species have been tested in
terms of phytoremediator power because they have par-
ticular traits appropriate for remediation. For instance,
maize and ambrosia were used for Pb [24], Thlaspi cae-
rulescens for Cd [25] and a fern that hyperaccumulates As
[26]. Other species assayed include pelargonium [27] and
sunflower [28] which can be cultivated as a monoculture,
or deep-rooted trees like poplar [29] and willow [30].
However none of them possess a complete set of charac-
teristics beneficial for phytoremediation, and they are
not (in most cases) promising commercial candidates.
An exhaustive screening of soils highly contaminated
by hazardous industrial waste in Eastern Spain was
carried out (manuscript submitted). Plants surviving at
these sites had developed resistance to metals such as
Cd, Pb, Zn, and Cu. Among them, N. glauca showed the
best physiological characteristics for phytoremediation
and was selected for subsequent gene transfer. The most
interesting features of this plant are: powerful root
system able to trap metals from deep soils, low nutrient
requirements, resistance to drought and heavy metals,
adaptation to wide geographic areas (continental
American, Australia, and parts of Europe), high bio-
mass (approximately 10 times more than Brassica jun-
cea), harvestability, easy vegetative multiplication,
biotechnologically handful, and repulsion of herbivores,
preventing its entry into the food chain.
For the first time a gene, different to the bacterial
virulence gene virF, (active in mediating T-DNA transfer
from Agrobacterium to plant cells), is introduced to
N. glauca [31]. The inserted gene (TaPCS1) is a wheat
PC synthase, capable to confer Cd tolerance upon yeast
[17]. When overexpressed in N. glauca, this gene in-
creased its tolerance to Pb. Root growth increased
drastically in transformed plants and leaves were higher
and greener (Fig. 1A). Interestingly, this rise in tolerance
is not correlated with an increased metal-exclusion ca-
pability, rather TaPCS1 enhanced expression triggered
by a higher lead transport (around 200%) to the root
tissue and to the aerial parts (near 150%), for plants
grown in contaminated soil (Fig. 2). Seedlings overex-
pressing TaPCS1 displayed higher tolerance to Cd as
well (Fig. 3). Since the main objective of this work was to
study Pb tolerance, the mine soil selected contained high
levels of this metal, but a low Cd concentration (Table
1). To clarify whether this increased tolerance is related
to a higher Cd absorption, pot experiments with highly
Cd contaminated soils have been initiated. The impor-
tance of these findings rests in the fact that the wheat
gene is capable of multiplying the metal tolerance in a
wild type plant, potentially exceptional for phytoreme-
diation. Furthermore, the experiment was performed in
a mine waste-contaminated soil (containing 1572 ppm of
Pb and 2602 ppm of Zn; Table 1), thus minimizing the
need to extrapolate results from laboratory to field.
So far biotechnology has been focused mainly on B.
juncea (which is related to Arabidopsis thaliana) over-
expressing genes reported to be key in both, cell redox
balance and phytochelatin synthesis, such as c-glutam-
ylcysteine synthetase [31]. The use of TaPCS1 is based
on three fundamental aspects: its effectiveness for Cd
has been demonstrated already in S. cerevisiae [17], it is
a gene from another plant (not bacterial); its function
and regulation are basically known [18]. This improved
metal tolerance might be a first step towards engineering
hyperaccumulation in this fast-growing, high biomass
plant species. Many more advances must be achieved,
for instance it would be advisable to control the repro-
ductive system of these genetically modified plants, and
therefore to limit the propagation beyond the zone to be
remediated. Interestingly, N. glauca is a species having
an additional value since it is commonly cultivated as an
ornamental plant which makes it a plant candidate to be
utilized in public gardens, reducing the lead contami-
nation (product of gasoline combustion) in the streets
and roads of many countries. Today, even populations
in the most remote areas of the Southern Hemisphere
have blood lead levels 50 times greater than the natural
background level, while the current US level of concern
exceeds it by 600-fold [32]. For that reason, environ-
mental departments of public administrations and pri-
vate companies have demonstrated interest in the
management of the plant obtained in this work.
444 C. Gisbert et al. / Biochemical and Biophysical Research Communications 303 (2003) 440–445

6. Acknowledgments
We thank Professor Julian Schroeder for TaPCS1 gene cession.
This work has been carried out in collaboration with Spanish private
companies COPUZOL and CAJA RURAL VALENCIA. Funds were
provided by the European Union (F.E.D.E.R., 1FD97-1469-C04-01)
and Spanish Ministry of Science and Technology. We thank also
Francisco Segura Head of Area of the Department of Environmental
Quality (‘‘Conselleria de Medio Ambiente’’) of the Council of the
Valencian Community and Alejandro Ribes of this same entity for
their important cooperation. We also thank Dr. Vicente Pall
a
as for
helpful suggestions and discussion, and specially to Dr. Carmen
Gonz
a
alez for critical reading of the manuscript and helpful comments.
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C. Gisbert et al. / Biochemical and Biophysical Research Communications 303 (2003) 440–445 445