# Patent

## Title
Biological contamination-removal method

## Abstract

The invention relates to a transgenic wild type plant species from Nicotiana glauca transformed with the phytochelatin synthase TaPCS gene from wheat for the phytoremediation of soils contaminated with heavy metals and other pollutants.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention is of application in the metal elimination that contaminates soils and aquatic zones.
2. State of the Art
At present, the state of the art provides alternative ways of solving the problem. The most habitual are also the costliest and they base on, fundamentally chemical and physical methods, as they are: the committal of the contaminated soils with inert materials, the cleanliness “in situ” with the machines and chemical suitable material, the storage as poisonous residues in the places regulated for it (with correspondent license), etc.
In the United States there have been carried out experiences of recovery of the contaminated soils, using natural plants, of the type of the corn, tomato, sunflower, Brassica juncea, etc., and trees principally willow and black poplar. Most of these pioneering experiences has been realized by the American army moved by the desire to recover soils been contaminated with the metallic garbage that provoke the bullets that are in use in the tests of shot. These metals contain fundamentally lead.
Also another type of experiences have been performed in New Jersey. It was carried out a selection of cultures of Brassica juncea (Brassica juncea (L) Czem) with aptitude to accumulate the elected metals; it was used enmendants of the soil or chelants substances. Later they allowed them to glow, and to be harvest, and the contain in metals have been analyzed. In these two cases, the used techniques have been fundamentally: treatment of contaminated soils, and regeneration by metal accumulation in the cultivated plants. The “U.S. Environmental Protection Agency”, also acquaintance as EPA, has been the pioneering institution in the study of these processes of phytoremediation. Together with the company “Phytotech” was the manager of realizing the mentioned process of recovery of the contaminated soils in New Jersey. In the experiments in which the objective has been the recovering of munition, as the one mentioned above, it has participated actively the engineers' body of the American army (Army Corps of Engineers). The company Phytotech is one of pioneers in the whole world. In Spain there exist references of ELECNOR, which it applies technologies of bioremediation patented by the North American company ECS.
Some examples of vegetable used material:

    
    
        hybrid trees of black poplar have been in use for extracting nickel, cadmium and zinc of contaminated soils (University of Georgia, USA, “Savannah River Ecology Laboratory”).
        In the department of energy of Ashtabula, Ohio, plants of sunflower have been in use for remediation of soils and waters contaminated by uranium.
    
    


Some examples of important patents are the following ones:

    
    
        U.S. Patent. Phytorecovery of metals using seedlings. U.S. Pat. No. 911,655, Aug. 15, 1997, Phytotech, Inc. (Monmouth Junction, N.J.).
        U.S. Patent. Method of using pelargonium sp. as hyperaccumulators for remediating contaminated soil. U.S. Pat. No. 185,797, Nov. 4, 1998, University of Guelph (Guelph, Calif.).
        U.S. Patent. Methods for removing pollutants from contaminated soil materials with a fern plant. U.S. Pat. No. 546,941, Apr. 11, 2000, University of Florida (Gainesville, Fla.). The invention describes the use of a variety that is less extensive geographically that N glauca. It is a question of a fern that basically accumulates enormous quantities of arsenic. Methods are described to carry out the decontamination using one or different varieties of ferns. The use of this type of plants has fundamentally the disadvantage of which there is located in very humid habitats which limits his use. Besides the need to receive very much water contribution can have negative effects for the soil where they grow since, on having contributed big quantities of water, this one can displease the metals at deeper levels or spread them.
    
    


Since it parts with the information contributed in the exposed patents, the work it has centred on processes of bioremediation, especially using hyperaccumulators (those plants that possess an extraordinary capacity of absorption of metals, and of accumulation in his biomass).
The phytoremediation consists basically of the use of plants or of vegetable material, to decontaminate soils with high concentrations of harmful elements. Recently a review has been realized, about the topic, by two Spanish investigators, in the publication: “Phytoextraction: to cost-effective plant-based technology for the removal of metals from the environment”. Carlos Garbisu, Itziar Alkorta. Bioresource Technology 77 (2001) 229-236. Where the state of the investigation is described in phytoremediation, topcoat being based on the concrete aspect of the phytoextraction.
One key article (review), for the importance of the works realized by the authors are the followings:

    Chaney, R. L., Malik, M., Li, Y. M., Brown, S. L., Brewer, E. P., Angle, J. S., Baker, A. J. M., 1997. Phytoremediation of soil metals. Current Opin. Biotechnol. 8, 279-284.
    Salt, D. E., Smith, R. D., Raskin, I., 1997. Phytoremediation of soil metals. Current Opin. Biotechnol. 8, 279-284. This is an article of reference for the punctual contribution and

Corn and buckthorn are two species that accumulate lead. (Huang, J. W., Cunningham, S. D., 1996. Lead phytoextraction: species variation in lead uptake). As for the cadmium, Salix viminalis, Thlaspi caerulescens and Alyssum murale seem to be the best species (SALIX EXPERT PHYTOEXTRACTOR, Maria Greger, Dept of Botany, Stockholm University, S-106 91 Stockholm, Sweden).



Concerning to the molecular biology of metal absorption, fundamentally researchers have worked with plants of the genre of Brassica, and among them especially Brassica juncea. In these type of plants genetic transformations have been realized by means of skills of biotechnology. Also they have been carried out in another type of vegetable varieties as black poplar and certainly Arabidopsis. Regarding the genes that have been in use for decontaminating heavy metals, it is necessary to distinguish basically three families: metallothioneins, phytochelatins, and genes that regulate the processes of oxidation-reduction. It is necessary to emphasize the pioneering work realized with bacterial genes that are specialized in the absorption of mercury, which marked a milestone in this field: Rugh, C. L., Wilde, H. D., Stack N. M., Thompson, D. M., Summers, A. O., Meagher, R. B., 1996. “Mercuric ion reduction in transgenic Arabidopsis thaliana plants expressing to modified bacterial mere gene”. Proc. Natl. Acad. Sci. USA 93, 3182-3187.
The most important works in this field, apart the mentioned one, have been carried out by Schroeder, Rea, and Therry. During the same year, Schroeder studied for the first time the enzyme produced by the gene TaPCS1, which is a phytochelatin of wheat. (The EMBO Journal Vol. 18 Not 12 pp. 3325-3333, 1999. “Tolerance to toxic metals by to gene family of phytochelatin synthases from plants and yeast”. Stephan Clemens, Eugene J. Kim, Dieter Neumann and Julian I. Schroeder). The second one cloned, the homolog phytochelatin of Arabidopsis (Proc. Natl. Acad. Sci. USA, Vol. 96, pp. 7110-7115, June 1999). “AtPCS1, to phytochelatin synthase from Arabidopsis: Isolation and in vitro reconstitution”. Olena K. Vatamaniuk, Stéphane Mari, Yu-Ping Lu, and Philip A. Rea). And the third one transformed in several occasions, Brassica juncea with genes that increase the metal absorption (“Cadmium Tolerance and Accumulation in Indian Mustard Is Enhanced by Overexpressing γ-Glutamylcysteine Synthetase”. Yong Liang Zhu, Elizabeth A. H. Pilon-Smits, Alice S. Tarun, Stefan U. Weber, Lise Jouanin, and Norman Terry. Plant Physiology, December 1999, Vol. 121, pp. 1169-1177).
SUMMARY OF THE INVENTION
When a place is contaminated with heavy metals, it can be the origin of a serious problem of environmental health and even of human health. The metals can happen to be dissolved in the water of rivers or source of water nearby, and also they can happen to be absorbed by plants or trees, giving place at its entry in the nourishing chain. Even, not happening any of these two processes, the metal can be accessible to any alive being that accidentally manipulates the waters or contaminated soils. Sometimes, there takes place such a serious problem of pollution (as it happened with the company Boliden, in the Aznalcollar region in the year 99), that the metals devastate the flora and fauna that they find to its step. It emerges therefore, a technical problem that consists of the manipulation of these contaminated places, either in order that they are not a danger for the environment, or in order that they are returned to his natural state before being contaminated.
The present invention provides a procedure to decontaminate a media of growth contaminated with metals, using individuals of the plant species Nicotiana glauca, and Nicotiana glauca modified genetically (for inclusion of the gene TaPCS1), during a period of sufficient time in order that the metals are absorbed by the roots and accumulated in the different tissues of the plant (root, stem, leaves). Being the metals of a group consisting of lead, arsenic, cadmium, copper, mercury, iron, chromium, uranium, nickel and zinc.
The proposed procedure uses the plant species Nicotiana glauca, chosen of a group of vegetable surviving species in a contaminated area, located in Valencia city.
At present, the state of the art provides alternative ways of solving the problem. The most habitual are also the costliest and they base on fundamentally chemical and physical methods, since they are: the committal of the soils contaminated with inert materials, the cleanliness “in situ” with the machines and chemical suitable material, the storage as poisonous residues in the places adapted for it (with license), etc. All these skills have the big disadvantage, of their high cost, in addition to which in any cases it is not possible to carry out the finished cleanliness. The committal, it does not look like a real solution to the problem, but rather a postponement of the same one in the time. The same might be said, of the storage of the soils contaminated in the places conditioned for it.
The techniques that use hyperaccumulator plants have the great disadvantage of which these plants possess some characteristics that dissuade them as solution, namely: low biomass, adjustment to a very concrete habitat, normally only accumulate a specific metal while they do not resist a set of diverse metals, and have a short cycle of life. What is looked actually, is that the plants absorb the metal pollutant (or the set of metals pollutants), and that they transport it to the parts able to be collected. Later this material would be harvested and it would arrange suitably or would burn itself (also it might study other alternatives).
In the current state of the art, since it has been mentioned previously, it has been elected another type of plants different from the hyperaccumulators, among them it is necessary to stand out on every Brassica juncea. Later, on these plants there have introduced genes that they qualify the plant species to a better response. In general, the use of the biotechnology to solve this type of problems of pollution, has the clear disadvantage of the enormous consumption of time that is needed to solve the problem, and that besides the process of decontamination has to be carried out almost always “in situ”. Nevertheless, the phytoremediation (disciplines that use vegetable material to decontaminate contaminated places), it is always a much less costly procedure in terms of money.
In the present invention, one manages to improve the state of the art previously developed. In this way:

    
    
        1.—Wild chosen flora: it is adapted to specific climatology and edaphology
        2.—It is very competitive (“weeds”) in “normal” situations and of metal excess.
        3.—Adapted to soils of low nutritional and water content (they resist water and nutritional stress).
        4.—Improved natural capacity. It means that it is probable that they are individuals modified genetically with regard to those who grow in not contaminated zones.
        5.—Improved capacity by means of genetic modification. There has been introduced a gene of wheat, which level of expression is permanently increased. The result is a major capacity of absorption of heavy metals, as Pb, Cd, etc.
    
    


The point one, it does not mean that these plants are not a transportable technology to other points of the world geography. Probably the opposite will happen, since one the advantages of the used plant species is that it survives habitually in extensive geographical areas of the world. This way for example, although it is original of the south and central part of the American continent, it spreads also over all United States and Canada, the whole Australian continent, and part of Europe and Japan. Besides, this plant species reproduces by cuttings, it replicates with facility in the field, provides abundant seeds, has aptitude to banish animals (which is a guarantee of his difficulty to enter the feed chain), is in use as ornamental plant in the United States and Canada, and all that together with the familiar beneficial characteristics for phytoremediation as they are: deep root system, high biomass, and adjustment to soils nutritionally low and of high pollution for metals. Another additional, not despicable advantage exists still, and the fact is that the technology of transformation of genes to vegetable species is limited to a limited group of plant species, among them the plant species that the investigators more have worked is Nicotiana tabaccum. Therefore, Nicotiana glauca, might benefit from the whole scientific technological undivided assets that has developed for Nicotiana tabaccum. 
In addition to the importance that has the vegetable species that has been chosen to be biotechnologically modified, it is necessary to emphasize the importance of the gene that has been transferred. The gene TaPCS1, it was studied in depth demonstrating a great specialization and yield in the absorption of lead and cadmium in yeast (used like model system). It was the first phytochelatin synthase that was studied, and it belongs to one vegetable species that is cultivated habitually in the exterior of the laboratories (it does not happen equally with Arabidopsis), what it could mean a better capacity of adjustment to the external environment. This gene had never been transformed into plant previously, hence the innovation and importance of this fact.
The importance of the role played by the phytochelatins in the detoxification and homeostasis of heavy metals, is still today an object of discussion. Recent works have shed some light in the molecular base of this process and also in the role of the phytochelatins and the phytochelatin synthases in the process of metal accumulation. Nevertheless part from the described investigation very recently it is incoherent with previous works and it needs an urgent clarification.
Phytoremediation and Metal-Binding Peptides
In the last decades the concern for the global Earth environment has impulse a major research focus in decontamination. The phytoremediation is an emerging and low cost technology that utilizes plants to remove, transform or stabilize toxic chemicals located in water, sediments or soils. Physiological studies indicate that heavy metal tolerance is one of the prerequisites of heavy metal hyperaccumulation in plants (Kramer et al., 1997; Raskin et al., 1997). Phytoremediation of heavy metals more likely require genetic engineering metal tolerance in selected wild plant species, which are fast-growing, of high biomass, and tolerant to a wide range of environmental conditions.
Living organisms have developed molecular processes that allow cells the capability to detoxify (in some particular concentration ranges) heavy metals. In eukaryotic cells, these mechanisms include mainly sequestration and chelation by specific ligands. Among these kind of molecules most of researchers interest has been focused in the last years in two of them: metallothioneins and phytochelatins. Metallothioneins are ubiquitous low molecular weight proteins and polypeptides of extremely high metal and sulfur content (Kägi J. H. and Schaffer A., 1988). Phytochelatins (PCs) are thiolate peptides with the primary structure (γ-Glu-Cys)n-Gly, (where n=2-11) non-translationally synthesized from glutathione (Grill, E. et al. (1989). PCs have been identified in a wide variety of plant species including monocots, di-cots, gymnosperms and algae (Rauser, 1999).
GSH is Substrate of Phytochelatin Synthase.
PCs are synthesized from Glutathione (GSH). GSH is synthesised in a two-step pathway (FIG. 1) catalysed by γ-glutamylcysteine synthetase (γ-ECs) and glutathione synthetase (GS); requires energy since both are ATP-dependent enzymes. The level of GSH decreases on the induction of PCs in plant cell cultures and the exposure of whole plants or plant cell cultures to an inhibitor of GSH biosynthesis, buthionine sulfoximine (BSO), also inhibits the induction of PCs and/or confers hypersensitivity to metal ions. Furthermore, in cell cultures the effects of BSO can be reversed by the addition of GSH evidencing a marked interaction between GSH and phytochelatin biosynthesis regulation.
The Key Role of PCs in Heavy Metal Tolerance is Discussed
What is the role played by PCs in heavy metal (HM) tolerance and accumulation? Do they are essential instruments in engineering phytoremediation? Do they play a basic role in plants that emerge more tolerant by natural selection under HM pressure? Most significant recent advances in the understanding of the general role played by PCs comes from molecular genetic studies using different plant and fungi model systems. Specially for the differential metal tolerance observed among naturally occurring selected plants, resistant and sensitive ecotypes.
Negative Evidence
Some researchers think that previous studies of phytochelatin production in response to Cd and Cu demonstrated that PCs are not responsible for metal tolerance although they are indeed involved in the detoxification process. This affirmation relays upon the fact that root tips of Cd-tolerant plants of Silene vulgaris exhibit a lower rate of phytochelatin production accompanied by a lower rate of longer chain phytochelatin synthesis than those of Cd-sensitive plants Silene vulgaris (De Knecht, et al. 1994). Regarding their role in naturally selected plants that have increased HM accumulation, some researchers believe that the investigation of tolerant Silene vulgaris plants from the copper mining dump shows that PCs are not responsible for the development of the heavy metal tolerant phenotypes. This is based in the fact that Cd- and Cu-complexes disappear in the roots of water cultures of Silene vulgaris between 7 and 14 days after heavy metal exposition. Although the binding of HM ions to PCs exists it seems to play only a transient role in the heavy metal detoxification mechanism of this plant species (Leopold, et al. 1999). Similar findings have been reported for the hyperaccumulator Thlaspi caerulescens and the related non-accumulator T. arvense. Total PC levels found in the hyperaccumulator were generally lower respect to the non-accumulator, despite correspondingly higher metal concentrations. However again, similarly to Silene vulgaris, PCs were produced by both species in response to Cd, and phytochelatin levels showed a similar positive correlation with Cd-concentration in leaf and root tissues (Ebbs, et al. 2002). What are the molecular bases of PCs enzymatic regulation for this apparent absence of relevance in HM tolerance? Although Cd-tolerant Silene vulgaris plants produce three times less PCs than Cd-sensitive ones, when are exposed to external supply of Cd, neither the PCS activity nor PC degradation accounts by for the difference in PCs concentration (De Knecht, et al. 1995). Therefore, all these evidences may suggest a lack of importance for the PCs role in Silene vulgaris and Thlaspi caerulescens respect to Cd tolerance. Consequently, from all this information some researchers have concluded that differential synthesis of PCs is not involved in producing differential metal tolerance. However it also can be argued that PCs concentration is three times higher in Cd-sensitive Silene vulgaris plants after external Cd supply respect to the non sensitive, therefore whether they really play a key role or not in these sensitive plants should be questioned.
Very recently a set of evidences against the possible central role of PCs in HM detoxification have been presented. When Arabidopsis thaliana PCS gene (AtPCS1) was overexpressed with the goal of increasing PC synthesis and therefore metal accumulation, transgenic lines showed paradoxically hypersensitivity to Cd. However, this hypersensitivity disappeared when GSH was supplemented in the medium. Consequently, it is argued that Cd hypersensitivity seems due to the toxicity of PCs, as they existed at supraoptimal levels when compared with GSH levels (Lee, S. et al. 2003).
Positive Evidence
The first evidence comes from two basic facts: plant cells when exposed to HM rapidly accumulate PCs and PCs production is activated by HM ions (Grill et al., 1989). When the activity of the partially purified enzyme was determined in vitro, the enzyme was active only in the presence of HM ions (being Cd the best activator). When the activity was determined in vivo (in both intact plants and plant cell cultures), HM also induced PC biosynthesis (Rauser, 1995). Lack and restoration of function has been also an important source of evidences. For example, it was reported that phytochelatin-deficient Saccharomyces pombe and Arabidopsis thaliana mutants are hypersensitive to Cd. Furthermore, AtPCS1 was able to suppress the Cd-sensitive phenotype in Brewer's yeast (Vatamaniuk, et al., 1999). Stronger evidence however, has come from a wheat cDNA, TaPCS1, whose expression in S. cerevisiae results in a dramatic increase in Cd tolerance (Clemens, et al., 1999). This work demonstrated that TaPCS1 expression led to an augment in Cd2− accumulation inside the yeast cells even at concentrations that do not affect the growth. More important, this group showed that heterologous expression of PCS genes is sufficient to confer enhanced metal tolerance. Performing growth assays with S. cerevisiae cells expressing AtPCS1 and SpPCS (S. pombe homolog), they have demonstrated that phytochelatin synthesis alone can significantly increase cellular Cd tolerance. Besides, when wheat roots were investigated for TaPCS1 expression, RT-PCR experiments indicated that this PCS expression is constitutive and enhanced by Cd, consistent with the reported constitutive activity of PCS (Grill, et al., 1989) in roots and stems (Chen, et al., 1997) and coherent with the suggested requirement for organisms to express metal tolerance genes constitutively (Zenk, 1996). Therefore PCS activity is regulated transcriptionally and post-transcriptionally by HM. Are these evidences strong enough to answer the question of whether PCs are essential for HM tolerance? There are now more observations supporting the idea of a key role of PCs in HM tolerance. Very recently the overexpression of gene TaPCS1 in Nicotiana glauca selected in a contaminated environment increases Cd tolerance and furthermore lead accumulation. Roots growth was improved drastically (near 160%) and leaves were bigger and greener in the transformed plants respect to wild type, in the presence of 50 μM Cd and even 0.8 mM lead. TaPCS1 transformed plants are able to growth in mining soils having as high as 1572 ppm of lead (2602 ppm of Zn), and accumulating twice lead level than wild type plants. TaPCS1 enhanced expression triggered a higher lead transport (around 200% accumulation) to the root tissue and to the aerial parts (near 150%). Furthermore this improved metal tolerance might be a first step towards engineering hyperaccumulation in this fast-growing, high biomass plant species (Gisbert, et al., 2003). All these positive evidences together strongly suggest that indeed PCs play a role of physiological and ecological relevance in this plant species and many other organisms beyond the manifest role of PCs in HM detoxification.
PCs are Basic Instruments in HM Tolerance, at Least in Some Organisms
A set of evidences has questioned the role played by PCs in HM detoxification. Differential Cd tolerance in S. vulgaris in tolerant and sensitive ecotypes is not affected by differential PC production per se. Increased Cd tolerance does not result from an increased accumulation of PCs, from a faster synthesis of longer PCs (forming more stable complexes than shorter forms), or from an increased incorporation of sulfide into PC-Cd complexes in the roots (which likely increases the stability and the potential amount of metal bound per unit of PC-SH). However, tolerant plants reach the same PC concentration as sensitive plants when exposed to higher Cd concentrations. Since the difference between the amounts of PCs induced neither is caused by a difference in the specific PCS activity, nor the rate of PC breakdown, it might then result from a lower Cd concentration in the cytosol caused by a faster transport of PC-Cd complexes, across the tonoplast into the vacuole (Salt, and Rauser, 1995). It also is possible, that tolerant plants contains a higher proportion of acid-soluble Cd, which is not bound to PCs or another complex in addition to PC-Cd is into the vacuole, or that the Cd ion itself is transported via the tonoplast antiporter activity (Salt, and Wagner 1993). Same arguments might likely explain the similar PC concentrations for T. caerulescens and T arvensis. Nevertheless it is interesting to note that the accumulator T. caerulescens produces a positive Cd gradient across the plant from roots to leaves (1000-3000 ppm) while T arvensis develops a higher but negative Cd gradient (7500-1000 ppm). Consequently, and following the same accumulation pattern in both Thlaspi species, shoot Cd and PC levels are higher in absolute values for T. caerulescens, and accordingly PCS showed a less rate of saturation for this ecotype in leaves. Consistently, in this tissue a higher ratio of Cd to γ-GluCys subunits has been observed for T. caerulescens as if Cd were preferentially sequestered as high molecular weight complexes (Ebbs, S. et al., 2002). Although these arguments might also contribute to understand the hypersensitivity reported by AtPCS1 overexpression in Arabidopsis, evidences are clearly contradictories with previously reported works (Vatamaniuk, et al. 1999; Clemens, et al., 1999). Perhaps, nonspecific protein-protein or others unknown interactions caused by the modification of the AtPCS1 C-terminal region by the FLAG epitope (DYKDDDL) used for protein recognition, may contribute to the observed Cd hypersensitivity in lines overexpressing AtPCS1 (Lee, et al. 2003). The C-terminal domain clearly has some role in PCS activity since the cad1-5 mutant synthesizes a truncated polypeptide predicted to lack nine of the 10 Cys residues in this C-terminal domain, producing an altered phenotype. It is likely that this domain acts as a local sensor by binding heavy metal ions (presumably via the multiple Cys residues, but possibly also others) and bringing them into contact with the activation site in the catalytic domain (Cobbett, C. S., 2000). Therefore, a possible negative interference of AtPCS1 C-terminal FLAG region with PC homeostasis should not be discarded. Beyond of what be the possible explanation for this Cd hypersensitivity, the facts are that after addition of a plant-specific phytochelatin chemically synthesized and, more important, after AtPCS1 expression in mammalian cells, the transfected cells exhibited production of plant-specific PC and higher resistance to Cd2+ (Takagi, et al., 2003). It is known that mammalian cells can not synthesize PC because of their lack of PCS.
The work reported specially by Clemens et al., 1999, Tagaki et al., 2003 and, very recently, by Gisbert et al., 2003 have presented solid and consistent evidences for a primary role of PCs in HM tolerance and detoxification in a wide range of organisms. Perhaps negative evidences accumulated might possibly indicate that PCs are essential but not the solitary essential component of the more complex mechanisms developed by organisms to detoxify HM. Consequently other basic elements, when increased or more effective, might reduce the need of a significant augment of PCs and PCS concentrations in such specific cases.
REFERENCES FOR THIS PARAGRAPH


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    Kägi J. H. and Schaffer A. (1988) Biochemistry of metallothionein. Biochemistry 27, 8509-8515
    Grill, E. et al. (1989) Phytochelatins, the heavy-metal-binding peptides of plants, are synthesized from glutathione by a specific γ-glutamylcysteine dipeptidyl transpeptidase (phytochelatin synthase). Proc. Natl Acad. Sci. USA 86, 6838-6842
    Rauser, W. E. (1999) Structure and function of metal chelators produced by plants; The case for organic acids, amino acids, phytin and metallothioneins. Cell. Biochem. Biophys. 31, 19-48
    De Knecht, J. A. et al. (1994) Phytochelatins in Cadmium-Sensitive and Cadmium-Tolerant Silene vulgaris. Plant Physiol. 104, 255-261
    Leopold, I. et al. (1999) Phytochelatins and heavy metal tolerance. Phytochemistry 50, 1323-1328
    Ebbs, S. et al. (2002) Phytochelatin synthesis is not responsible for Cd tolerance in the Zn/Cd hyperaccumulator Thlaspi caerulescens. Planta 214, 635-640
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    Rauser, W. E. (1995) Phytochelatins and related peptides: structure, biosynthesis, and function. Plant Physiol. 109, 1141-1149
    Vatamaniuk O. K. et al. (1999) AtPCS1, a phytochelatin synthase from Arabidopsis: isolation and in vitro reconstitution. Proc Natl Acad Sci USA 96, 7110-7115
    Clemens, S. et al., (1999) Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast. EMBO J. 18, 3325-3333
    Chen, J. et al. (1997) Characterization of phytochelatin synthase from tomato. Physiol. Plant. 101, 165-172
    Zenk, M. H. (1996) Heavy metal detoxification in higher plants—a review. Gene, 179, 21-30
    Gisbert, C. et al. (2003). A plant genetically modified that accumulates Pb is specially promising for phytoremediation. Biochem. Biophys. Res. Commun. 303, 440-445
    Salt, D. E. and Rauser, W. E. (1995) MgATP-dependent transport of phytochelatins across the tonoplast of oat roots. Plant Physiol 107, 1293-1301
    Salt D. E., Wagner G. J. (1993) Transport of Cd in tonoplast vesicles from oat roots. Evidence for a Cd/H antiport activity. J Biol Chem 268, 12297-12302
    Cobbett, C. S. (2000) Phytochelatins and their roles in heavy metal detoxification. Plant Physiol. 123, 825-832
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BRIEF DESCRIPTION OF THE DRAWING
FIG. 1. is a chart showing PC biosynthesis in plants and yeast. Glutathione (GSH) is a substrate for phytochelatin synthase (PCS). Cd is a positive effector of γ-ECS, Glutathione synthase (GS) and PCS. γ-ECS is also feedback regulated by GSH. Both γ-ECS and CS are ATP dependent. Cd is a positive effector of CS. The phytochelatin is bound to Cd and forms a complex PC-Cd. The complex is transported inside the vacuole by a transporter type YCF1.



DETAILED DESCRIPTION
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 BamHI-Ecl136II sites of plasmid pBI121. The new construct (pBITaPCS1) was electroporated into Agrobacterium tumefaciens strain C58C1RifR Rif (Van Larebeke et al. 1974).
Nicotiana glauca leaf explants were infected with A. tumefaciens after two days of preculture on organogenic medium NB2510 [MS salts (Murashige y Skoog, 1962) including Gamborg B5 vitamins (DUCHEFA), 3% sucrose, 2.5 μg mL−1 naphthalene acetic acid (NAA), 1 μg mL−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 10 min. After 1 day of cocultivation, explants were transferred to selection medium NB2510 containing 100 μg mL−1 kanamycin and 350 μg mL−1 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 μg mL−1 indol acetic acid or 0.2 μg mL−1 NAA, 1% sucrose, 100 μg mL−1, and 0.7% plant agar).

## Claims

1. A plant species transformed for the phytoremediation of a soil contaminated with heavy metals, the plant species comprising: a progenitor plant of Nicotiana glauca genetically transformed with the gene TaPCS1, wherein the progenitor plant of Nicotiana glauca is obtained from polluted soils.

2. The plant species according to claim 1, wherein the heavy metals are selected from the group consisting of cadmium, lithium, arsenic, copper, chromium, silver, zinc, lead, mercury, nickel, cobalt, tin, ruthenium, rhodium, uranium, iridium, polonium uranium, cesium, boron, osmium, niobium, tantalum, gold, antimony, bismuth, indium, scandium, titanium, palladium, zirconium, platinum, technetium, rhenium, lantanids and actinids.

3. The plant species according to claim 1, wherein the TaPCS gene is in a binary Ti vector pBI121.

4. The plant species according to claim 1, wherein the plant is a wild type plant and is being used for the same soil or for other polluted soil after creation of the genetically transformed plant.

5. The plant species according to claim 2, wherein the heavy metals in the soil are selected from the group consisting of cadmium and lead.

6. A plant species transformed for the phytoremediation of a soil contaminated with heavy metals; the plant species comprising: a progenitor plant of Nicotiana glauca genetically transformed with the gene TaPCS1, wherein the progenitor plant of Nicotiana glauca is obtained from polluted soils, and wherein the contaminated soil comprises at least 1,500 ppm of lead and/or at least 2,500 ppm of zinc.

7. A method for phytoremediating a soil contaminated with heavy metals, the method comprising the steps of:
1) obtaining a progenitor Nicotiana glauca plant from a polluted soil;
2) transforming the Nicotiana glauca plant with the gene TaPCS1; and
3) transferring the transformed Nicotiana glauca plant to the contaminated soil.

8. The method of claim 7, wherein the contaminated soil comprises at least 1,500 ppm of lead and/or at least 2,500 ppm of zinc.