生物修复转基因植物

r m inia pollution Plants are autotrophic organisms capable of using sunlight duction [5,6]. However, phytoremediation also suffers Update olism of toxic volatile pollutants. In doing so, they have delivered a technology that is likely to lead to the wider contaminants from soil, phytoremediation involves differ- ent processes, such as enzymatic degradation, that poten- tially lead to contaminant detoxification (Figure 1) [3–5]. However, despite great promise, rather slow removal rates and potential accumulation of toxic compounds within plants might have limited the application of phytoremedia- tion [1]. In a recent study, Doty et al. [6] have developed transgenic poplars with an enhanced uptake and metab- bacterial and mammalian degradative enzymes can there- fore be used to complement the metabolic capabilities of plants [1]. Historically, transgenic plants for phytoreme- diation were first developed in an effort to improve heavy metal tolerance; for example, tobacco plants (Nicotiana tabacum) expressing a yeast metallothionein gene for higher tolerance to cadmium, orArabidopsis thaliana over- expressing a mercuric ion reductase gene for higher toler- ance to mercury [8,9]. The first attempts to transform plants for phytoremediation of organic compounds tar- carbons and explosives [3,4]. Beyond the removal of mals possess the enzymatic machinery necessary to achieve a complete mineralization of organic molecules; and carbon dioxide as sources of energy and carbon. How- ever, plants rely on the root system to take up water and othernutrients, suchasnitrogenandminerals, fromsoil and groundwater. As a side effect, plants also absorb a diversity of natural and man-made toxic compounds for which they have developed diverse detoxification mechanisms [1]. Pol- lutant-degrading enzymes inplants probably originate from natural defense systems against the variety of allelochem- icals released by competing organisms, including microbes, insects and other plants [2]. From this viewpoint, plants can be seen as natural, solar-powered pump-and-treat systems for cleaning up contaminated environments, leading to the concept of phytoremediation [3]. First developed for the removal of heavy metals from soil, the technology has since proven to be efficient for the treatment of organic com- pounds, including chlorinated solvents, polyaromatichydro- Research Focus Transgenic plants for phyto nature to clean up environ Benoit Van Aken Department of Civil and Environmental Engineering, West Virg Phytoremediation is the use of plants to clean up environmental pollution. However, detoxification of organic pollutants by plants is often slow, leading to the accumulation of toxic compounds that could be later released into the environment. A recent publication by Doty and colleagues describes the development of trans- genic poplars (Populus) overexpressing a mammalian cytochrome P450, a family of enzymes commonly involved in the metabolism of toxic compounds. The engineered plants showed enhanced performance with regards to the metabolism of trichloroethylene and the removal of a range of other toxic volatile organic pollu- tants, including vinyl chloride, carbon tetrachloride, chloroform and benzene. This work suggests that trans- genic plantsmight be able to contribute to thewider and safer application of phytoremediation. Introduction: phytoremediation – plants to clean up application of phytoremediation in the field. Corresponding author: Van Aken, B. (benoit.vanaken@mail.wvu.edu). from several limitations, amongwhich themost commonly evoked are the slow rate of removal, incomplete metab- olism and potential increase in bioavailability of toxic contaminants [1,3]. Indeed, in the absence of significant detoxification, parent compounds and toxic metabolites can accumulate inside plant tissues and eventually return to the soil or volatilize into the atmosphere. The recognition that plants can transform xenobiotic compounds emerged in the 1940s, when plants were shown to metabolise pesticides [7]. Since then, the development of genomics, proteomics and metabolomics has exposed the plant metabolism of many xenobiotic compounds [1]. Plants often use pathways and enzymes similar to those of mammals, which led to the ‘green liver’ concept (Figure 2) [7]. However, being autotrophic organisms, plants do not actually use organic compounds for their energy and carbon metabolism. As a consequence, they usually lack the catabolic enzymes necessary to achieve full mineralization of organic molecules, potentially result- ing in the accumulation of toxic metabolites [1]. Hence, the idea to enhance plant biodegradation by genetic transform- ation was developed, following a strategy similar to that used to develop transgenic crops [1,5]. Transgenic plants for phytoremediation Typically, transgenic plants exhibiting new or improved phenotypes are engineered by the overexpression and/or introduction of genes from other organisms, such as bac- teria or mammals. Being heterotrophs, bacteria and mam- emediation: helping ental pollution University, Morgantown, WV 26506, USA From polluted soils to ‘toxic plants’ In comparison with other clean-up technologies, phytoremediation has potentially many advantages, in- cluding low installation and maintenance costs, less dis- ruption of the environment and other beneficial side effects such as carbon sequestration and biofuel pro- geted explosives and halogenated organic compounds in tobacco plants [10,11]. 225 Figure 1. Phytoremediation involves several processes: pollutants in soil and groundwater can be taken up inside plant tissues (phytoextraction) or adsorbed to the roots es med Update Trends in Biotechnology Vol.26 No.5 However, although tobacco and A. thaliana are good laboratory models, their small stature might not be suitable for field applications. Hence, there is particular interest in the genetic transformation of poplar trees (Populus sp.), which are fast growing plants with high biomass – ideal attributes for phytoremediation. Plant transformation is usually performed using the ‘natural (rhizofiltration); pollutants inside plant tissues can be transformed by plant enzym pollutants in soil can be degraded by microbes in the root zone (rhizosphere biore genetic engineer’ Agrobacterium tumefaciens, a plant pathogen that has become the favorite vector for gene Figure 2. The three phases of the ‘green liver’ model. Hypothetical pathway representin TCE by oxidation to trichloroethanol; phase II, conjugation with a plant molecule; phas 226 transfer to plants [12]. However, A. tumefaciens-mediated transformation of forest trees is notoriously challenging, which explains why there have been only a few reports about the genetic modification of poplar plants [13]. Gull- ner et al. [14] developed the first transgenic poplars for phytoremediation. Their transgenic line was designed to treat chloroacetanilide herbicides by the overexpression of (phytotransformation) or can volatilize into the atmosphere (phytovolatilization); iation) or incorporated in soil material (phytostabilization) [3–5]. a gamma-glutamylcysteine synthetase, an enzyme involved in glutathione synthesis. g the metabolism of trichoroethylene (TCE) in plant tissues. Phase I, activation of e III, sequestration of the conjugate into the cell wall or within the vacuole [7]. Poplar trees overexpressing a mammalian the first report about genetic engineering of plants for hydrocarbons in transgenic plants containing mammalian Update Trends in Biotechnology Vol.26 No.5 cytochrome P450 Cytochrome P450s constitute a large enzyme superfamily commonly involved in the metabolism of toxic compounds. In 2000, Doty et al. [11] described the development of transgenic tobacco plants expressing a human cytochrome P450 and capable of metabolizing trichloroethylene (TCE) 640-fold faster than wild type plants. The same group later reported the introduction of a rabbit cytochrome P450 in transgenic hairy root cultures of Atropa belladonna, which also exhibited a faster metabolism of TCE [15]. In the current study, Doty et al. [6] described the genetic trans- formation of hybrid poplar plants (Populus tremula � Populus alba) overexpressing mammalian cytochrome P450 2E1 (CYP2E1). The engineered trees were capable of the enhanced metabolism of five volatile toxic compounds: TCE, vinyl chloride, carbon tetrachloride, chloroform and benzene. Among the different transgenic clones tested, the most efficient one, line 78, expressed CYP2E1 at a 3.7- to 4.6-fold higher level and exhibited the highest level of TCE metabolism (>100-fold higher than in non-transgenic controls). When cultivated in hydroponic solution spiked with toxic compounds, line 78 was capable of extracting �90% of TCE (compared with <3% extracted by non-transgenic controls), 99% of chloro- form (compared with 20% by controls) and 92–94% of carbon tetrachloride (compared with 20% by controls). Enhanced metabolism of organic pollutants in transgenic plants is associated with a faster uptake, which can be explained by a steeper concentration gradient inside plant tissues [10,11,16]. Transgenic plants were also shown to remove volatile compounds from contaminated air at a higher rate than non-transgenic controls: 79% of TCE (none removed by controls), 49% of vinyl chloride (com- pared with 29% by controls) and �40% of benzene (com- pared with 13% by controls). All the five compounds under study are listed as Priority Pollutants by the U.S. Environ- mental Protection Agency (EPA) (http://oaspub.epa.gov/) and figure in the top 50 of the 2005 Priority List of Hazardous Substances defined by the Comprehensive Environmental Response, Compensation and Liability Act (CERCLA) (http://www.atsdr.cdc.gov/). These com- pounds are as follows: TCE, the most prevalent soil pollu- tant found in the United States; vinyl chloride, a dead-end carcinogenic compound produced from TCE biodegrada- tion; carbon tetrachloride, a common toxic solvent also found inmany polluted soils; chloroform, a toxic by-product formed during water disinfection by chlorination; and benzene, a carcinogenic compound found in association with petroleum pollution. Conclusions Transgenic poplars (CYP2E1) enhance both the uptake and the metabolism of several toxic solvents and could therefore help to overcome a major limitation inherent to phytoremediation – namely, the threat that accumulated toxic compoundswould volatilize or otherwise contaminate the food chain. Although the study by Doty et al. [6] is not cytochrome P450 2E1. Proc. Natl. Acad. Sci. U.S.A. 97, 6287–6291 12 Gelvin, S.B. (2003) Agrobacterium-mediated plant transformation: the biology behind the ‘gene-jockeying’ tool. Microbiol. Mol. Biol. Rev. 67, 16–37 13 Han, K.H. et al. (2000) An Agrobacterium tumefaciens transformation protocol effective on a variety of cottonwood hybrids (genus Populus). Plant Cell Rep. 19, 315–320 14 Gullner, G. et al. (2001) Enhanced tolerance of transgenic poplar plants overexpressing gamma-glutamylcysteine synthetase towards chloroacetanilide herbicides. J. Exp. Bot. 52, 971–979 15 Banerjee, S. et al. (2002) Expression of functional mammalian P450 2E1 in hairy root cultures. Biotechnol. Bioeng. 77, 462–466 16 Trapp, S. and Karlson, U. (2001) Aspects of phytoremediation of organic pollutants. J. Soils & Sediments 1, 37–43 17 Hills, M.J. et al. (2007) Genetic use restriction technologies (GURTs): strategies to impede transgene movement. Trends Plant Sci. 12, 177– 183 0167-7799/$ – see front matter � 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2008.02.001 Available online 18 March 2008 phytoremediation applications, it constitutes a milestone in the field for several reasons: first, it is one of the very few studies describing the successful development of trans- genic poplars, which is technically challenging [13]; second, the technology is efficient for the treatment of several important organic pollutants likely to be found in mixture in the environment; and third, it constitutes the achieve- ment of a pioneer work initiated by the same group a decade ago. With federal regulations limiting the use of transgenic forest trees, further developments of phytore- mediation are likely to involve genetic use restriction technologies (GURTs) for controlling the dispersion of transgenes in the environment [17]. As for transgenic crops, risks inherent to genetically modified organisms have to be minimized and balanced with the increasing needs of an ever-expanding human population. References 1 Eapen, S. et al. (2007) Advances in development of transgenic plants for remediation of xenobiotic pollutants. Biotechnol. Adv. 25, 442–451 2 Singer, A. (2006) The chemical ecology of pollutants biodegradation. In Phytoremediation and Rhizoremediation: Theoretical Background (Mackova, M. et al., eds), pp. 5–21, Springer 3 Pilon-Smits, E. (2005) Phytoremediation. Annu. Rev. Plant Biol. 56, 15–39 4 Salt, D.E. et al. (1998) Phytoremediation. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 643–668 5 Dietz, A.C. and Schnoor, J.L. (2001) Advances in phytoremediation. Environ. Health Perspect. 109, 163–168 6 Doty, S.L. et al. (2007) Enhanced phytoremediation of volatile environmental pollutants with transgenic trees. Proc. Natl. Acad. Sci. U.S.A. 104, 16816–16821 7 Sandermann, H. (1994) Higher plant metabolism of xenobiotics: The ‘green liver’ concept. Pharmacogenetics 4, 225–241 8 Misra, S. and Gedamu, L. (1989) Heavy-metal tolerant transgenic Brassica napus L. and Nicotiana tabacum L. plants. Theor. Appl. Genet. 78, 161–168 9 Rugh, C.L. et al. (1996) Mercuric ion reduction and resistance in transgenic Arabidopsis thaliana plants expressing a modified bacterial merA gene. Proc. Natl. Acad. Sci. U.S.A. 93, 3182–3187 10 French, C.E. et al. (1999) Biodegradation of explosives by transgenic plants expressing pentaerythritol tetranitrate reductase. Nat. Biotechnol. 17, 491–494 11 Doty, S.L. et al. (2000) Enhanced metabolism of halogenated 227 Transgenic plants for phytoremediation: helping nature to clean up environmental pollution Introduction: phytoremediation - plants to clean up pollution From polluted soils to ‘toxic plants’ Transgenic plants for phytoremediation Poplar trees overexpressing a mammalian �cytochrome P450 Conclusions References