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Phytoremediation of selenium using transgenic plants

Elizabeth AH Pilon-Smits1 and Danika L LeDuc2
Selenium (Se) is a micronutrient for many organisms but also toxic at higher concentrations. Both selenium deciency and toxicity are serious problems worldwide. Owing to the similarity of selenium to sulfur, plants readily take up and assimilate selenate via sulfur transporters and enzymes and can even volatilize selenium. Selenium accumulating or volatilizing plants may be used for phytoremediation of selenium pollution and as fortied foods. Several transgenic approaches have been used successfully to further enhance plant selenium accumulation, tolerance, and volatilization: upregulation of genes involved in sulfur/selenium assimilation and volatilization, methylation of selenocysteine, and conversion of selenocysteine to elemental Se. Lab and eld trials with different transgenic plants have yielded promising results, showing up to ninefold higher levels of selenium accumulation and up to threefold faster volatilization rates.
Addresses 1 Biology Department, Colorado State University, Anatomy/Zoology Building, Fort Collins, CO 80523, United States 2 Department of Chemistry & Biochemistry, California State University, East Bay, 25800 Carlos Bee Boulevard, Hayward, CA 94542, United States Corresponding author: Pilon-Smits, Elizabeth AH (epsmits@lamar.colostate.edu) and LeDuc, Danika L (danika.leduc@csueastbay.edu)

weight basis, but while these plants grow signicantly better in the presence of Se, so far it has not been shown that they cannot complete their life cycle in the absence of Se [5,6]. While probably not essential for plants, Se is considered a benecial element since it can promote plant growth and antioxidant capacity in many species, as well as resistance to herbivores and pathogens (for reviews see [7,8]). While Se levels in most soils are very low, Se occurs naturally in certain Cretaceous shale sediments. Seleniferous soils can contain up to 100 mg/kg Se, and when these soils are cultivated, or when fossil fuels derived from these soils are used, toxic levels of Se may accumulate in the environment [9]. Selenium hyperaccumulator plants thrive on seleniferous soils, providing another portal for Se into the environment [8]. For animals, there is a narrow window between the amount of Se required as a nutrient and the amount that is toxic; as a consequence, both Se deciency and toxicity are common problems worldwide [10]. Plants may help alleviate both of these problems, either as a source of dietary Se or for phytoremediation of excess Se from the environment. Selenium-enriched diets have been shown to prevent different forms of cancer, viral infections including HIV, and male infertility (for a review see [11]). Plants have also been shown to be able to clean up agricultural and industrial wastewaters and soils [1214], owing to their capacity to not only take up and accumulate Se but also convert inorganic Se into volatile forms that are released into the atmosphere (Figure 1). To use plants more efciently for phytoremediation or as Se-fortied foods, it is helpful to know how plants take up and metabolize Se and identify the rate-limiting steps involved. Our current knowledge about plant Se metabolism is summarized in Figure 2 (for reviews, see [10,15]). Owing to its similarity to S, Se can make use of S transporters and metabolic pathways. Selenate, the predominant bioavailable form of Se in most soils, is taken up by plants, reduced to selenite, and then assimilated into the selenoamino acids, selenocysteine (SeCys), and selenomethionine (SeMet). Non-specic incorporation of these selenoamino acids into proteins, replacing Cys and Met, is toxic [5]. Plants have evolved several mechanisms to avoid this process. Firstly, methylation of SeCys and SeMet leads to accumulation of the non-protein amino acids methyl-SeCys (MetSeCys) and methylSeMet (MetSeMet), which can be safely accumulated. This detoxication mechanism is particularly important in Se hyperaccumulator species [16]. Secondly, MetSeCys or MetSeMet can be further metabolized to
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Current Opinion in Biotechnology 2009, 20:16 This review comes from a themed issue on Plant biotechnology Edited by Dave Dowling and Sharon Lofferty Doty

0958-1669/$ see front matter Published by Elsevier Ltd. DOI 10.1016/j.copbio.2009.02.001

Introduction
Selenium (Se) is an essential nutrient for many organisms, but also toxic at higher levels. As a nutrient, Se is an essential component of certain selenoproteins, some of which are important for scavenging free radicals and preventing cancer and infections [1]. The toxicity of Se at higher concentrations is thought to be mainly due to its chemical similarity to sulfur (S), leading to non-specic replacement of sulfur by Se in proteins and other sulfur compounds. While Se is essential for mammals, bacteria, and some green algae like Chlamydomonas, it has not been shown to be essential for higher plants [2,3,4]. It has been suggested that Se is essential for so-called hyperaccumulator plants, which can accumulate 0.11.5% Se on a dry
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Figure 1

volatile dimethylselenide (DMSe, in non-hyperaccumulators) or dimethyldiselenide (DMDSe, in hyperaccumulators) [6,17]. Finally, SeCys may be broken down into alanine and the relatively innocuous elemental Se [18]. This same enzyme activity plays an important role in organisms that require Se, where the elemental Se released from SeCys is specically incorporated into essential selenoproteins [19]. Using existing knowledge about rate-limiting steps in plant Se metabolism and Se detoxication mechanisms in tolerant species, several different plant genetic engineering strategies have been designed and used successfully to further enhance plant Se uptake, accumulation, volatilization, and tolerance. Below we review these various strategies.

Transgenic approaches to enhance Se tolerance, accumulation, and volatilization

Enhancing assimilation of selenate to selenocysteine

Movement and possible fates of selenium in plants. The predominant bioavailable form of Se in soils is selenate. After selenate is taken up by plants, it can be accumulated in the root and also translocated to the shoot. Inorganic selenate can be assimilated into selenocysteine and other forms of organic Se. Some forms of organic Se are volatile, and can be emitted by the plant. Plant accumulation and volatilization may be used in phytoremediation, or to create Se-fortified foods.

From plant physiological studies, the reduction of selenate to selenite appeared to be a rate-limiting step for the assimilation of selenate into organic Se: plants that were supplied with selenate accumulated mainly selenate while plants that were fed selenite accumulated mainly organic Se [20,21]. To overcome this apparent rate limitation in Se metabolism, the rst enzyme mediating selenate-to-selenite conversion, ATP sulfurylase (APS) from Arabidopsis thaliana was constitutively expressed in Brassica juncea (Indian mustard) [22]. The resulting APS transgenics accumulated an organic form of Se when

Figure 2

Overview of plant Se metabolism, showing the enzymes discussed here in italics. Metabolites with a green border are safe ways for the cell to accumulate Se, while compounds with a red border are unsafe sinks for Se. OAS: O-acetylserine; OPH: O-phosphohomoserine. Current Opinion in Biotechnology 2009, 20:16 www.sciencedirect.com

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supplied with selenate, in contrast to wild-type (untransformed) controls that accumulated selenate. Interestingly, the APS plants accumulated twofoldthreefold more Se than wild type. Perhaps because of their enhanced Se assimilation, the APS transgenics tolerated the accumulated Se better than wild type. Selenium volatilization rate was unaltered in the APS transgenics. From these studies, it can be concluded that ATP sulfurylase is involved in selenate reduction and is rate limiting for the assimilation of selenate to organic Se. The enhanced APS expression also appears to trigger selenate uptake and Se accumulation.
Enhancing the conversion of selenocysteine to volatile dimethylselenide

accumulated [16]. MetSeCys can be further metabolized to volatile, non-toxic DMDSe [17], and emitted into the atmosphere. The A. bisulcatus SMT enzyme has been overexpressed in two different host plants, A. thaliana and B. juncea [25,26]. In both species, SMT-overexpressing plants exposed to selenite successfully converted SeCys to MetSeCys [25 27]. The ability to convert SeCys to MetSeCys correlated with increased total Se accumulation in both species. SMT-expressing A. thaliana plants accumulated up to approximately 1000 mg Se/kg dry weight [25] and SMT-expressing B. juncea accumulated almost 4000 mg Se/kg dry weight [26]. The SMT-expressing plants accumulated higher total Se concentrations while at the same time better tolerating selenite and SeCys [25,26]. Additionally, SMT-expressing B. juncea had signicantly improved selenate tolerance compared with wild-type plants. The expression of SMT also resulted in increased rates of Se volatilization, seen in particular with A. thaliana grown on SeCys and B. juncea exposed to selenate, with more volatile Se produced in the form of DMDSe [26,28]. These experiments show that the SMT enzyme is a key enzyme for Se hyperaccumulation, conferring both enhanced Se tolerance and accumulation when expressed in non-hyperaccumulators.
Conversion of selenocysteine to elemental selenium

Since overexpression of APS did not enhance Se volatilization, another enzyme downstream in the Se assimilation pathway was apparently limiting for this process. Physiological volatilization studies pointed to the conversion of SeCys to SeMet, since volatilization from SeMet was much faster than from SeCys. On the basis of the kinetic properties of the enzymes involved in this conversion, the rst enzyme in the pathway from SeCys to SeMet, cystathionine gamma synthase (CgS), was hypothesized to be rate limiting and chosen for overexpression. Indeed, the resulting transgenic B. juncea plants, constitutively expressing the A. thaliana CgS enzyme, showed twofoldthreefold higher volatilization rates compared with untransformed plants [23]. The CgS transgenics accumulated 40% less Se in their tissues than the wild type, probably as a result of their enhanced volatilization. The CgS plants were also more tolerant than wild-type plants, perhaps because of their lower tissue Se levels. On the basis of these results, it can be concluded that CgS is involved in and rate limiting for Se volatilization.
Methylation of selenocysteine

The toxicity of Se is attributed largely to the misincorporation of SeCys in proteins, erroneously taking the place of Cys and disturbing protein structure and function [5]. Thus, plant Se tolerance may be enhanced by converting SeCys into other, less dangerous forms of Se, leaving less SeCys to compete with Cys for incorporation into proteins. The strategy of methylating SeCys is used by Se hyperaccumulators such as Astragalus bisulcatus, which thrive on seleniferous soils and accumulate up to 1.5% Se in their tissues (15 000 mg Se/kg dry weight). Hyperaccumulators are generally less suitable for phytoremediation or as fortied foods because they grow slowly and are not very palatable [24]. However, they may be a good source of genes to overexpress in fastgrowing, high biomass crop species. The key enzyme responsible for Se hyperaccumulation and tolerance in A. bisulcatus is SeCys methyltransferase (SMT). SMT confers tolerance to Se by specically methylating SeCys to MetSeCys, a non-protein amino acid that can be safely
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The strategy to precipitate accumulated Se as elemental Se is used as a detoxication mechanism by many bacteria [29]. In addition, the conversion of SeCys to elemental Se is a rst step toward incorporation of Se into selenoproteins in organisms that require Se [19]. Enzymes that specically convert SeCys into alanine and elemental Se are called SeCys lyases (SL). With the expectation that the specic breakdown of SeCys will prevent its misincorporation into proteins and thus enhance plant Se tolerance, a mouse SL enzyme was expressed in both A. thaliana and B. juncea [18,30]. In A. thaliana, the mouse SL was targeted to either the cytosol or the chloroplast, since both are locations where SeCys can interfere with protein translation. As hypothesized, plant expression of the mouse SL led to reduced Se incorporation into proteins in all types of transgenics. When expressed in the cytosol of A. thaliana, the mouse SL enhanced plant Se tolerance, as expected. However, when the mouse SL was expressed in the chloroplast of A. thaliana, Se tolerance was reduced. This may have been due to interference of the produced elemental Se with ironsulfur cluster formation, which uses elemental S and takes place in chloroplasts but not in the cytosol. All the transgenic SL plants showed enhanced Se accumulation, up to twofold compared with wild-type plants. When the mouse SL enzyme was expressed in A. thaliana, it was discovered serendipitously that the wild-type plants had SL activity as well. The native SL enzyme
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activity was shown to be partly chloroplast-localized, and this plastidic protein with SL activity was called CpNifS [18]. When this enzyme was overexpressed in the chloroplast of A. thaliana, the resulting CpNifS transgenics showed reduced incorporation of Se in proteins, twofold enhanced Se accumulation, and enhanced Se tolerance [31]. Thus, the results were comparable with those of the mouse SL overexpressors with the exception that, while expressed in the chloroplast, CpNifS conferred enhanced tolerance. Perhaps, being a native enzyme, it was subject to regulation at the protein level. Together, these results show that the specic breakdown of SeCys can effectively reduce incorporation of Se into proteins, and as long as the elemental Se does not interfere with cellular processes, this enhances Se tolerance. It also appears that introducing this new sink for Se in the cell induces upregulation of Se (and S) uptake and assimilation.
Double transgenics with enhanced selenium assimilation and selenocysteine methylation

would also be useful as fortied food, particularly if they accumulate MetSeCys since this form of Se is particularly anticarcinogenic [33]. The results described above were mostly obtained under controlled laboratory conditions, using Se-spiked agar or nutrient solution. To further assess their potential use for phytoremediation or as Se-fortied foods, the transgenics described above were tested for their capacity to extract Se from naturally seleniferous soil or from Se-contaminated sediment (phytoextraction). In a greenhouse pot experiment using naturally seleniferous soil, the APS transgenics accumulated threefold higher Se levels than wild-type B. juncea, while CgS transgenics contained 40% lower Se levels than wild type, both in agreement with the earlier laboratory results [34]. Plant biomass was the same for all plant types. In a eld experiment on Se-contaminated sediment in the San Joaquin Valley (CA, USA), the APS transgenics accumulated fourfold higher Se levels than wild-type B. juncea, similar to the laboratory and greenhouse results [35]. In another eld experiment on the same Se-polluted sediment, the cpSL and SMT transgenics showed twofold higher Se accumulation compared with wild-type B. juncea, in agreement with earlier laboratory experiments [36]. In both eld experiments, there were no signicant differences between the plant types with respect to biomass production. In conclusion, the results obtained from the different transgenics under controlled laboratory conditions were essentially the same as those obtained when using naturally seleniferous or Se-contaminated soils in greenhouse or eld. The various transgenics showed enhanced Se accumulation, volatilization, and/ or tolerance, all promising traits for use as Se-fortied foods or for phytoremediation.

Although the expression of SMT in B. juncea enhanced Se tolerance, accumulation, and volatilization [26], these effects were more substantial when the SMT-expressing plants were exposed to selenite relative to selenate. The reduction of selenate to selenite most probably provided a rate-limiting barrier to the production of SeCys, greatly diminishing any potential benet of overexpressing SMT. To overcome this rate limitation, double-transgenic plants were created that overexpress both APS and SMT [32] (APS SMT plants, obtained by crossing of APS and SMT transgenics). The APS SMT double transgenics accumulated almost eight times more MetSeCys than wild type and nearly twice as much as the SMT transgenics [32]. As for total Se, the APS SMT double transgenics accumulated up to nine times higher Se concentrations than wild type and signicantly more Se than either single transgenic line when treated with selenate concentrations between 200 and 500 micromolar [32]. This increase in internal Se concentration in the APS SMT transgenics did not result in decreased biomass production, so the Se accumulation capacity (concentration biomass) was successfully enhanced [32]. Thus, by combined overexpression of the enzymes APS and SMT, it has been possible to combine the ability of the APS enzyme to enhance selenate uptake and reduction to selenite and SeCys with the ability of the SMT enzyme to methylate the SeCys and thus to detoxify the increased pool of internal Se.

Future prospects for phytoremediation of selenium using transgenic plants

With the arrival of the genomic era, new genes and proteins will probably be discovered to promote Se tolerance, accumulation, and volatilization. Also, comparative studies of Se hyperaccumulators and related nonaccumulators or of Se-tolerant ecotypes and non-tolerant ecotypes of the same species may reveal new genes that upregulate Se uptake, (hyper)accumulation, and volatilization. Such newly discovered genes may be outside of the commonly studied area of sulfur metabolism. For instance, a Se-binding protein (SBP) homolog, when overexpressed in A. thaliana, enhanced tolerance to Se as well as cadmium [37]. Its function is so far unknown, but has been hypothesized to be similar to glutathione, binding excess toxins. Moreover, recent genetic and genomic studies [38,39,40] have identied new quantitative trait loci (QTL) and genes involved in Se tolerance. The plant hormones jasmonic acid (JA) and ethylene are emerging as important players in plant responses to Se tolerance, possibly via their inuence on S and Se assimilation.
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Testing the transgenics for enhanced Se phytoextraction from environmental soils in the greenhouse and in the eld
Transgenic plants with enhanced Se accumulation, tolerance and volatilization could potentially be used for phytoremediation. Plants with enhanced Se accumulation
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Further studies may reveal key control genes that trigger the cascade of responses that together provide Se tolerance and accumulation in model plants and hyperaccumulators. Such key genes could be the ultimate candidates for overexpression, producing the complete Se hyperaccumulator prole in high-biomass crop species. Before existing and future transgenics are used at a large scale in the eld for phytoremediation or as fortied foods, it is desirable to study the potential ecological implications of growing Se accumulating or volatilizing plants. How will the plant Se affect the local ecosystem? Is there any risk to pollinators or herbivores, and how is microbial activity affected? Studies so far indicate that plant Se deters a wide variety of invertebrate and vertebrate herbivores (for reviews see [8,41]), suggesting limited risk of plant Se to herbivores and for accumulation of Se in the food chain. In seleniferous habitats, however, Se-tolerant specialists appear to have evolved that thrive on high-Se plants and that may form a portal for Se into the local ecosystem. A Se-tolerant diamondback moth was found to feed on hyperaccumulator plants and to be in turn parasitized by a Se-tolerant parasitic wasp [42]. Both species contained around 250 mg/kg Se, potentially lethal levels for predators. The evolution of Se tolerance in the diamondback moth in the USA is particularly interesting because this species invaded this country only relatively recently (<100 years), showing that Se tolerance can evolve fairly quickly. Thus, while plant Se tends to deter herbivores, reducing the risk for bioaccumulation of Se in the food chain, growing Se-rich plants for long periods in the same area may lead to evolution of Setolerant herbivores that may form a portal for Se into the local ecosystem. Additional considerations for the use of transgenics for phytoremediation are the same as those involved with growing transgenics for other purposes and should also be evaluated and weighed against the risks of alternative remediation methods.

system, Chlamydomonas reinhardtii. EMBO J 2002, 21:3681-3693. 4.  Lobanov AV, Fomenko DE, Zhang Y, Sengupta A, Hateld DL, Gladyshev VN: Evolutionary dynamics of eukaryotic selenoproteomes: large selenoproteomes may associate with aquatic life and small with terrestrial life. Genome Biol 2007, 8:R198. The authors compared the selenoproteome of several model eukaryotes, that is, proteins that contain selenocysteine as an essential component. On the basis of their study, selenoproteins originated at the base of the eukaryotic domain, and are still present in many bacteria, mammals, and several green algae. However, essential selenium metabolism appears to have gotten lost in higher plants as well as in fungi, nematodes, and insects. In these groups cysteine tends to have taken over the role of selenocysteine. 5. Brown T, Shrift A: Exclusion of selenium from proteins in selenium-tolerant Astragalus species. Plant Physiol 1981, 67:1951-1953. Anderson JW: Selenium interactions in sulfur metabolism. In Sulfur nutrition and assimilation in higher plants - Regulatory, agricultural and environmental aspects. Edited by De Kok LJ. SPB Academic Publishing; 1993:49-60. Hartikainen H: Biogeochemistry of selenium and its impact on food chain quality and human health. J Trace Elem Med Biol 2005, 18:309-318.

6.

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Quinn CF, Galeas ML, Freeman JL, Pilon-Smits EAH: Selenium: deterrence, toxicity, and adaptation. Integr Environ Assess Man 2007, 3:460-462. A review is given on ecological and evolutionary aspects of selenium hyperaccumulation in plants, as well as ecological implications of growing selenium-fortied crops. While selenium has been shown to deter herbivory and to be toxic to herbivores and pathogens, some herbivores in seleniferous areas have also been shown to have evolved tolerance to this mode of elemental defense. 9. Ohlendorf HM, Hoffman DJ, Salki MK, Aldrich TW: Embryonic mortality and abnormalities of aquatic birds: apparent impacts of selenium from irrigation drain water. Sci Total Environ 1986, 52:49-63.

10. Terry N, Zayed A, de Souza M, Tarun A: Selenium in higher plants. Annu Rev Plant Physiol Plant Mol Biol 2000, 51:401-432. 11. Ellis DR, Salt DE: Plants, selenium and human health. Curr Opin Plant Biol 2003, 6:273-279. uelos GS, Meek DW: Accumulation of selenium in plants 12. Ban grown on selenium-treated soil. J Environ Qual 1990, 19:772-777. 13. Hansen D, Duda PJ, Zayed A, Terry N: Selenium removal by constructed wetlands: role of biological volatilization. Environ Sci Technol 1998, 32:591-597. 14. Lin Z-Q, Terry N: Selenium removal by constructed wetlands: quantitative importance of biological volatilization in the treatment of selenium-laden agricultural drainage water. Environ Sci Technol 2003, 37:606-615. 15. Sors TG, Ellis DR, Salt DE: Selenium uptake, translocation, assimilation and metabolic fate in plants. Photosynth Res 2005, 86:373-389. ck A: On the mechanism of selenium tolerance in 16. Neuhierl B, Bo selenium-accumulating plants. Purication and characterization of a specic selenocysteine methyltransferase from cultured cells of Astragalus bisulcatus. Eur J Biochem 1996, 239:235-238. 17. Lewis B, Johnson C, Delwiche C: Release of volatile selenium compounds by plants: collection procedures and preliminary observations. J Agric Food Chem 1966, 14:638-664. 18. Pilon-Smits EAH, Garifullina GF, Abdel-Ghany SE, Kato S-I, Mihara H, Hale KL, Burkhead JL, Esaki N, Kurihara T, Pilon M: Characterization of a NifS-like chloroplast protein from Arabidopsis thalianaImplications for its role in sulfur and selenium metabolism. Plant Physiol 2002, 130:1309-1318. 19. Mihara H, Kato S, Lacourciere G, Stadtman TC, Kennedy RAJD, Kurihara T, Tokumoto U, Takahashi Y, Esaki N: The iscS gene is Current Opinion in Biotechnology 2009, 20:16

Acknowledgements
The writing of this manuscript was supported by National Science Foundation grant # IOS-0817748 to EAHPS. We thank Wiebke Tapken for creative design of gures.

References and recommended reading

Papers of particular interest published within the period of review have been highlighted as:  of special interest  of outstanding interest 1. 2. Stadtman TC: Selenocysteine. Annu Rev Biochem 1996, 65:83-100. Fu L-H, Wang X-F, Eyal Y, She Y-M, Donald LJ, Standing KG, BenHayyim G: A selenoprotein in the plant kingdom: mass spectrometry conrms that an opal codon (UGA) encodes selenocysteine in Chlamydomonas reinhardtii glutathione peroxidase. J Biol Chem 2002, 277:25983-25991. Novoselov SV, Rao M, Onoshko NV, Zhi H, Kryukov GV, Xiang Y, Weeks DP, Hateld DL, Gladyshev VN: Selenoproteins and selenocysteine insertion system in the model plant

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essential for the biosynthesis of 2-selenouridine in tRNA and the selenocysteine-containing formate dehydrogenase. Proc Natl Acad Sci U S A 2002, 99:6679-6683. 20. de Souza MP, Pilon-Smits EAH, Lytle CM, Hwang S, Tai JC, Honma TSU, Yeh L, Terry N: Rate-limiting steps in selenium volatilization by Brassica juncea. Plant Physiol 1998, 117:1487-1494. 21. Zayed A, Lytle CM, Terry N: Accumulation and volatilization of different chemical species of selenium by plants. Planta 1998, 206:284-292. 22. Pilon-Smits EAH, Hwang SB, Lytle CM, Zhu YL, Tai JC, Bravo RC, Leustek T, Terry N: Overexpression of ATP sulfurylase in Brassica juncea leads to increased selenate uptake, reduction and tolerance. Plant Physiol 1999, 119:123-132. 23. van Huysen T, Abdel-Ghany S, Hale KL, LeDuc D, Terry N, PilonSmits EAH: Overexpression of cystathionine-g-synthase in Indian mustard enhances selenium volatilization. Planta 2003, 218:71-78. 24. Cunningham S, Shann J, Crowley D, Anderson T: Phytoremediation of contaminated water and soil. In Phytoremediation of Soil and Water Contaminants (ACS Symp Ser No 664). Edited by Kruger E, Anderson T, Coats J. Washington, D.C.: Am Chem Soc; 1997:2-17. 25. Ellis DR, Sors TG, Brunk DG, Albrecht C, Orser C, Lahner B, Wood KV, Harris HH, Pickering IJ, Salt DE: Production of Semethylselenocysteine in transgenic plants expressing selenocysteine methyltransferase. BMC Plant Biol 2004, 4:1. n M, Meija J, Malit MF, Wu CP, 26. LeDuc DL, Tarun AS, Montes-Bayo AbdelSamie M, Chiang C-Y, Tagmount A, de Souza MP et al.: Overexpression of selenocysteine methyltransferase in Arabidopsis and Indian mustard increases selenium tolerance and accumulation. Plant Physiol 2004, 135:377-383. n M, LeDuc DL, Terry N, Caruso J: Selenium 27. Montes-Bayo speciation in wild-type and genetically modied Se accumulating plants with HPLC separation and ICP-MS/ESMS detection. J Anal At Spectrom 2002, 17:872-879. n M, LeDuc DL, Terry N, Caruso J: 28. Meija J, Montes-Bayo Simultaneous monitoring of volatile selenium and sulfur species from Se accumulating plants (wild-type and genetically modied) by GC-MS and GC-ICP-MS using SPME for sample introduction. Anal Chem 2002, 74:5837-5844. 29. Garbisu C, Ishii T, Leighton T, Buchanan BB: Bacterial reduction of selenite to elemental selenium. Chem Geol 1996, 132:199-204. 30. Garifullina GF, Owen JD, Lindblom S-D, Tufan H, Pilon M, PilonSmits EAH: Expression of a mouse selenocysteine lyase in Brassica juncea chloroplasts affects selenium tolerance and accumulation. Physiol Plant 2003, 118:538-544. 31. Van Hoewyk D, Garifullina GF, Ackley AR, Abdel-Ghany SE, Marcus MA, Fakra S, Ishiyama K, Inoue E, Pilon M, Takahashi H, Pilon-Smits EAH: Overexpression of AtCpNifS enhances selenium tolerance and accumulation in Arabidopsis. Plant Physiol 2005, 139:1518-1528.

n M, Wu CP, 32. LeDuc DL, AbdelSamie M, Montes-Bayo Reisinger SJ, Terry N: Overexpressing both ATP sulfurylase and selenocysteine methyltransferase enhances selenium phytoremediation traits in Indian mustard. Environ Pollut 2006, 144:70-76. 33. Unni E, Koul D, Alfred Yung W-K, Sinha R: Semethylselenocysteine inhibits phosphatidylinositol 3-kinase activity of mouse mammary epithelial tumor cells in vitro. Breast Cancer Res 2005, 7:R699-R707. 34. van Huysen T, Terry N, Pilon-Smits EAH: Exploring the selenium phytoremediation potential of transgenic Brassica juncea overexpressing ATP sulfurylase or cystathionine-g-synthase. Int J Phytorem 2004, 6:111-118. uelos G, Terry N, LeDuc DL, Pilon-Smits EAH, Mackey B: Field 35. Ban trial of transgenic Indian mustard plants shows enhanced phytoremediation of selenium-contaminated sediment. Environ Sci Technol 2005, 39:1771-1777. uelos G, LeDuc DL, Pilon-Smits EAH, Tagmount A, Terry N: 36. Ban Transgenic Indian mustard overexpressing selenocysteine lyase or selenocysteine methyltransferase exhibit enhanced potential for selenium phytoremediation under eld conditions. Environ Sci Technol 2007, 41:599-605. 37. Agalou A, Roussis A, Spaink HP: The Arabidopsis seleniumbinding protein confers tolerance to toxic levels of selenium. Funct Plant Biol 2005, 32:881-890. 38. Zhang L-H, Byrne PF, Pilon-Smits EAH: Mapping quantitative trait loci associated with selenate tolerance in Arabidopsis thaliana. New Phytol 2006, 170:33-42. 39. Tamaoki M, Freeman JL, Pilon-Smits EAH: Cooperative ethylene  and jasmonic acid signaling regulates selenite resistance in Arabidopsis thaliana. Plant Physiol 2008, 146:1219-1230. Genomic and biochemical comparison of two A. thaliana concessions that differ in selenite tolerance showed that the tolerant accession had higher expression levels of genes involved in jasmonic acid (JA) and ethylene production and response, including sulfur assimilation-related genes. Its tissue levels of JA and ethylene were also higher. Mutants in the tolerant accession that are impaired in JA/ethylene production showed reduced selenite tolerance, while external supply of JA or ethylene to the sensitive accession enhanced tolerance. Thus, JA and ethylene appear to be important hormones for plant Se tolerance; they may confer tolerance by upregulation of S assimilation. 40. Van Hoewyk D, Takahashi H, Hess A, Tamaoki M, PilonSmits EAH: Transcriptome and biochemical analyses give insights into selenium-stress responses and selenium tolerance mechanisms in Arabidopsis. Physiol Plant 2008, 132:236-253. 41. Pilon-Smits EAH, Freeman JL: Environmental cleanup using plants: biotechnological advances and ecological considerations. Front Ecol Environ 2006, 4:203-210. 42. Freeman JL, Quinn CF, Marcus MA, Fakra S, Pilon-Smits EAH: Selenium-tolerant diamondback moth disarms hyperaccumulator plant defense. Current Biol 2006, 16:2181-2192.

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