A plant genetically modified that accumulates Pb is especiallypromising for phytoremediation

Carmina Gisbert,a Roc Ros,b Antonio De Haro,c David J. Walker,d M. Pilar Bernal,d

Ram�oon Serrano,a and Juan Navarro-Avi~nn�ooa,*

a Departamento de Biolog�ııa del estr�ees, IBMCP, CSIC, Camino de Vera s.n., Valencia 46022, Spainb Departamento de Fisiolog�ııa Vegetal, Universidad de Valencia, Campus de Burjasot, Burjasot, Valencia 46100, Spain

c Departamento de Agronom�ııa y Mejora Gen�eetica Vegetal, Instituto de Agricultura Sostenible, CSIC, Alameda del Obispo s.n., C�oordoba 14080, Spaind Departamento de Conservaci�oon de Tierra y Agua y Administraci�oon de Residuos Org�aanicos, Centro de Edafolog�ııa y Biolog�ııa 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.

� 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 revolutionand 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 kindof 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~nn�oo).

0006-291X/03/$ - see front matter � 2003 Elsevier Science (USA). All rights reserved.

doi:10.1016/S0006-291X(03)00349-8

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 couldresult 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 manyyears 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 showinginteresting 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,

2:5lgmL�1 naphthalene acetic acid (NAA), 1lgmL�1 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 10min.

After 1 day of cocultivation, explants were transferred to selection

medium NB2510 containing 100lgmL�1 kanamycin and 350lgmL�1

carbenicillin. Two months after infection, shoots were individually

removed from the call using explants and transferred to bottles con-

taining 30ml of B1 medium (MS salts including Gamborg B5 vita-

mins, 0:3lgmL�1 indol acetic acid or 0:2lgmL�1 NAA, 1% sucrose,

100lgmL�1, and 0.7% plant agar). Cultures were incubated in a

growth chamber (24–26 �C, 16 h light at 120lmolm�2 s�1 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

71lmolm�2 s�1 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 10min and

subsequently in sterile deionized water for 10 and 15min. Fifty ster-

ilized seeds were sown in squared plates on medium PM (1.69mM

CaðNO3Þ2H2O; 16.8mM KNO3; 10gL�1 sucrose; 7gL�1 agar; pH 5).

After sterilization by filtration of a solution of CdCl2 and PbðNO3Þ2 we

obtained plates with 0, 0.15, 0.20, or 0.25mM 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 120lmolm�2 s�1 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 6gL�1 agar, MS salts [12], and

10gL�1 sucrose at pH 5.7 buffered with 0:25gL�1 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 10min, 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 0.6M.

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 144gkg�1.

The soil is a Xeric Torriorthent [15]. It has total concentrations of Pb

and Zn (1572 and 2602mgkg�1, 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 <4mm for pot experiments and to <2mm 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

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. Forinstance, 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, suitabilityfor 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 Valenciacity, 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 themetal 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

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 grownduring 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 geneTaPCS1was 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 greatestdifferences 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 m�1) 2.21 —

CaCO3 (%) 28.0 —

CEC (cmolc kg�1) 10.6 —

OM (%) 0.65 —

Organic-C (gkg�1) 3.8 —

Total-N (gkg�1) 0.3 —

Fe (gkg�1) 53.1 —

Mn (gkg�1) 1.6 —

Cu (mgkg�1)a 42 50–140

Pb (mgkg�1)a 1572 50–300

Zn (mgkg�1)a 2602 150–300

Ni (mgkg�1)a <2.0 30–75

Cd (mgkg�1)a <0.2 1–3

Cr (mgkg�1)a <2.0 100–150

a European Union limits (mgkg�1) 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

1572mgkg�1 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 50mM 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

lines were obtained at a 30 lM Cd (Fig. 3). This confirmsthe 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 onas 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], andare 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 contaminatedby 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 nutrientrequirements, 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 bacterialvirulence 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 toleranceis 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 highlevels 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 ina 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 basedon 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 populationsin 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

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�aas for

helpful suggestions and discussion, and specially to Dr. Carmen

Gonz�aalez for critical reading of the manuscript and helpful comments.

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(letter to the editor), New Engl. J. Med. 326 (1992) 1293–1294.

C. Gisbert et al. / Biochemical and Biophysical Research Communications 303 (2003) 440–445 445