BBRC

Biochemical and Biophysical Research Communications 303 (2003) 440445 www.elsevier.com/locate/ybbrc

A plant genetically modied 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 n Serrano,a and Juan Navarro-Avin ~o a,* Ramo
a s, IBMCP, CSIC, Camino de Vera s.n., Valencia 46022, Spain Departamento de Biolog a del estre Departamento de Fisiolog a Vegetal, Universidad de Valencia, Campus de Burjasot, Burjasot, Valencia 46100, Spain c Departamento de Agronom a y Mejora Gene tica Vegetal, Instituto de Agricultura Sostenible, CSIC, Alameda del Obispo s.n., Co rdoba 14080, Spain d n de Tierra y Agua y Administracio n de Residuos Orga nicos, Centro de Edafolog Departamento de Conservacio a y Biolog a Aplicada del Segura, CSIC, Campus de Espinardo, Apartado 4195, Murcia 30080, Spain b

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 modication, 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 eorts. 2003 Elsevier Science (USA). All rights reserved.
Keywords: Cadmium; Hyperaccumulators; Lead; Nicotiana glauca; Phytochelatin synthase; Bioremediation

Toxic metal pollution of the biosphere has accelerated rapidly since the onset of the industrial revolution and heavy metal toxicity poses major environmental and health problems. It is estimated that cleanup of hazardous 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 polluted environments. The primary sources of this metal include mining and smelting of metalliferous ores, burning of leaded gasoline, disposal of municipal sewage, 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 longterm persistence in the environment. For instance, Pb one of the more persistent metals, was estimated to have a soil retention time of 1505000 years [3]. Also, the
* Corresponding author. Fax: +34-96-387-78-59. ~o ). E-mail address: jpavinyo@upvnet.upv.es (J. Navarro-Avin

average biological half-life of cadmium has been estimated to be about 18 years [4]. The use of biological materials to cleanup heavy metal contaminated soils has been focused on as an efcient and aordable form of bioremediation. Phytoremediation is an emerging and low cost technology that utilizes plants to remove, transform, or stabilize contaminants located in water, sediments, or soils. Vegetation growing in contaminated sites has developed a mechanism of tolerance to the inadequate environments, therefore a well adapted ora, tolerant to edafoclimatic conditions can be a basic instrument for phytoremediation [5,6]. Dierent 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 accumulating 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

0006-291X/03/$ - see front matter 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00349-8

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contaminated with toxic metals and accumulating extraordinary 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 dierent hyperaccumulator species have been identied [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 o, in landll 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 metalaccumulating 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 rst step in engineering metal tolerance in a selected wild plant species, Nicotiana glauca R. Graham, which is fastgrowing, of high biomass, and tolerant of a wide range of environmental conditions. This involved transformation of the plants with the wheat PC synthase gene (TaPCS1), and subsequent determination of their heavy metal accumulation and tolerance, specically Pb and Cd.

2:5 lg mL1 naphthalene acetic acid (NAA), 1 lg mL1 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 100 lg mL1 kanamycin and 350 lg mL1 carbenicillin. Two months after infection, shoots were individually removed from the call using explants and transferred to bottles containing 30 ml of B1 medium (MS salts including Gamborg B5 vitamins, 0:3 lg mL1 indol acetic acid or 0:2 lg mL1 NAA, 1% sucrose, 100 lg mL1 , and 0.7% plant agar). Cultures were incubated in a growth chamber (2426 C, 16 h light at 120 lmol m2 s1 photon ux density; Grolux, Sylvania, uorescent tubes). Regenerated plantlets were acclimatized in pots (25 cm diameter) with a mixture of peat and vermiculite (3:1) in a growth incubator (2527 C, 16 h light at 71 lmol m2 s1 photon ux density, and 62% relative humidity) and then transferred to the greenhouse. Progenies were obtained from those transgenic plants by selng 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 sterilized seeds were sown in squared plates on medium PM (1.69 mM CaNO3 2 H2 O; 16.8 mM KNO3 ; 10 g L1 sucrose; 7 g L1 agar; pH 5). After sterilization by ltration of a solution of CdCl2 and PbNO3 2 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 PbNO3 2 . Medium pH was adjusted to 5. After 9 days at 25 C, 16 h light at 120 lmol m2 s1 photon ux density (Grolux, Sylvania, uorescent 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 6 g L1 agar, MS salts [12], and 10 g L1 sucrose at pH 5.7 buered with 0:25 g L1 MES (2-[N-morpholino]ethanesulfonic acid). Ten days after (when the rst 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 PbZn 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 144 g kg1 . The soil is a Xeric Torriorthent [15]. It has total concentrations of Pb and Zn (1572 and 2602 mg kg1 , 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 56 days, and sieved to <4 mm for pot experiments and to <2 mm for analysis. Total heavy metals were extracted by nitric acidperchloric 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].

Materials and methods

Agrobacterium strain, plants transformation, and culture conditions The binary Ti vector pBI121 (Clontech) was used for transformation. 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 pYESTaPCS1. The plasmid was digested with XhoI and converted to blunt ends with the DNA polymerase I (Klenow fragment). Afterwards, 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 BamHIEcl136II sites of plasmid pBI121. The new construct (pBITaPCS1) was electroporated into Agrobacterium tumefaciens strain C58C1Rif R 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,

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Results Selection of a natural plant species specially good for phytoremediation Soils highly contaminated by heavy metals over several decades were studied, searching for plant species particularly useful for phytoremediation of this area and others with similar agro climatic characteristics (manuscript submitted). Heavy metal concentrations were determined for soils and plant tissues collected from contaminated sites. Most of the analyzed soils were located in the metropolitan area of Valencia city (manuscript 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 classied following a set of priorities: low nutrient requirements, heavy metal tolerance, high biomass, low water requirements, ease of vegetative propagation, 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) fullled all the requirements and was selected for further studies. Interestingly this plant species spans extended geographic areas such as Europe, Australia, and continental America, which favors its possible application for bioremediation 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 elevated 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 nally achieved (see Materials and methods) by incorporating and overexpressing the gene TaPCS1, which encodes a wheat phytochelatin synthase, shown to increase cadmium tolerance in yeast [17]. Phytochelatins have the structure (l-GluCys)n-Gly, where n has been reported as being as high as 11, but is generally in the range 25 [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 modied 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 modied plants (TaPCS1 insertion was conrmed 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 modied lines (1 and 2), grown for 10 days in agar media containing no added Pb (upper line) or 0.8 mM PbNO3 2 (lower line). Root lengths (right panel) are displayed in mm (w: wild type, 1, 2, and 3 are lines including TaPCS1). Twenty-ve 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.

C. Gisbert et al. / Biochemical and Biophysical Research Communications 303 (2003) 440445 Table 1 Characteristics of the soil (M4) used in pot experiments Characteristic pH EC (dS m1 ) CaCO3 (%) CEC (cmolc kg1 ) OM (%) Organic-C (g kg1 ) Total-N (g kg1 ) Fe (g kg1 ) Mn (g kg1 ) Cu (mg kg1 )a Pb (mg kg1 )a Zn (mg kg1 )a Ni (mg kg1 )a Cd (mg kg1 )a Cr (mg kg1 )a
a

443

M4 7.70 2.21 28.0 10.6 0.65 3.8 0.3 53.1 1.6 42 1572 2602 <2.0 <0.2 <2.0
1

EUa 67

50140 50300 150300 3075 13 100150

Fig. 2. Percent of increment in internal Pb concentration. Seedlings of wild type (w) and modied (m) plants growing in mine soil containing 1572 mg kg1 Pb, were analyzed after separation of the aerial parts and the roots. Results are the average of three dierent experiments. Standard deviation is less than 10%.

European Union limits (mg kg ) for agricultural soils [14].

presumption that overexpression of TaPCS1 might increase lead tolerance and absorption in N. glauca, the plant species chosen for bioremediation purposes. Genetically modied 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 eect on plant roots developed supplying 50 lM CdCl2 to N. glauca. Seedlings overexpressing the gene TaPCS1 had longer roots (near 160%) and higher and greener leaves than unmodied plants, resembling the behavior of seedlings supplemented with PbNO3 2 . As expected CdCl2 incites higher toxicity than PbNO3 2 for the reason that the concentration range used to test out the seed-tolerance to Cd, comes near three orders of magnitude less. The greatest dierences in growth between wild type and transformed

concentrations tested roots were longer than those of wild type plants, even when they were shorter in the Pb medium (line 3), displaying maximum dierences at 0.8 mM PbNO3 2 . 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 modied 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 modied plants (Fig. 2). Besides, lead absorption reaches up by about a 85% (82:68 4) increment in the roots of plants containing the gene TaPCS1 (981 ppm in ashes). These results conrm the initial

Fig. 3. Seedling growth in control and high-Cd media. Left panel: wild type (w) and modied 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).

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lines were obtained at a 30 lM Cd (Fig. 3). This conrms 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 ecient and aordable form of bioremediation [21,22]. The conventional technologies used for in situ and ex situ remediation are typically expensive and destructive [23]. Hyperaccumulators such as Thlaspi and Alyssum species [5] emerge, at rst, as an obvious way to approach the question, however, this type of plant species have important limitations. First, hyperaccumulators are usually specic for one particular metal [5], and are adapted to precise climate and soil conditions (not really transferable). Furthermore, they cannot be managed 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 biotechnologically traits that make the modied 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 screenselected wild type plant species. The studies have focused on two of the most worrisome contaminants: Pb and Cd. Up to now, dierent plant species have been tested in terms of phytoremediator power because they have particular traits appropriate for remediation. For instance, maize and ambrosia were used for Pb [24], Thlaspi caerulescens for Cd [25] and a fern that hyperaccumulates As [26]. Other species assayed include pelargonium [27] and sunower [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 characteristics benecial 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 biomass (approximately 10 times more than Brassica juncea), harvestability, easy vegetative multiplication, biotechnologically handful, and repulsion of herbivores, preventing its entry into the food chain.

For the rst time a gene, dierent 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 increased 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 capability, 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 overexpressing 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 importance of these ndings rests in the fact that the wheat gene is capable of multiplying the metal tolerance in a wild type plant, potentially exceptional for phytoremediation. 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 eld. So far biotechnology has been focused mainly on B. juncea (which is related to Arabidopsis thaliana) overexpressing genes reported to be key in both, cell redox balance and phytochelatin synthesis, such as c-glutamylcysteine synthetase [31]. The use of TaPCS1 is based on three fundamental aspects: its eectiveness 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 rst 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 reproductive system of these genetically modied 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 contamination (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, environmental departments of public administrations and private companies have demonstrated interest in the management of the plant obtained in this work.

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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 as for helpful suggestions and discussion, and specially to Dr. Carmen Gonz alez for critical reading of the manuscript and helpful comments. [15]

[16] [17]

[18]

[19]

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Further Reading
[1] A.R. Flegal, D.R. Smith, Lead levels in preindustrial humans (letter to the editor), New Engl. J. Med. 326 (1992) 12931294.