生物修复与净化土壤

h populations for a genetic analysis of naturally selected metal human health. In wealthy industrialized countries con- tamination is often highly localized, and the pressure to of the growth period, plant biomass is harvested, dried or incinerated, and the contaminant-enriched material is use contaminated land and water for agricultural food production or for human consumption, respectively, is minimal. However, soil and water contamination is wide- spread in Eastern Europe, and is increasingly recognized as dramatic in large parts of the developing world, pri- marily in India [2] and China [3]. deposited in a special dump or added into a smelter. The energy gained from burning of the biomass could support the profitability of the technology, if the resultant fumes can be cleaned appropriately. For phytoextraction to be worthwhile, the dry biomass or the ash derived from above ground tissues of a phytoremediator crop should www.sciencedirect.com Current Opinion in Biotechnology 2005, 16:133–141 hyperaccumulation in plants, and from comprehensive ionomics data – multi-element concentration profiles from a large number of Arabidopsis mutants. Addresses Max Planck Institute of Molecular Plant Physiology, Am Mu¨hlenberg 1, D-14476 Golm, Germany Corresponding author: Kra¨mer, Ute (kraemer@mpimp-golm.mpg.de) Current Opinion in Biotechnology 2005, 16:133–141 This review comes from a themed issue on Plant biotechnology Edited by Dirk Inze´ Available online 2nd March 2005 0958-1669/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2005.02.006 Introduction Pollution with metals and xenobiotics is a global envir- onmental problem that has resulted from mining, indus- trial, agricultural and military practices [1]. Many pollutants accumulate in the food chain and threaten Phytoremediation: novel approac Ute Kra¨mer Environmental pollution with metals and xenobiotics is a global problem, and the development of phytoremediation technologies for the plant-based clean-up of contaminated soils is therefore of significant interest. Phytoremediation technologies are currently available for only a small subset of pollution problems, such as arsenic. Arsenic removal employs naturally selected hyperaccumulator ferns, which accumulate very high concentrations of arsenic specifically in above- ground tissues. Elegant two-gene transgenic approaches have been designed for the development of mercury or arsenic phytoremediation technologies. In a plant that naturally hyperaccumulates zinc in leaves, approximately ten key metal homeostasis genes are expressed at very high levels. This outlines the extent of change in gene activities needed in the engineering of transgenic plants for soil clean-up. Further analysis and discovery of genes for phytoremediation will benefit from the recent development of segregating es to cleaning up polluted soils Although the avoidance of pollution should certainly be the primary objective, this principle has not generally been followed in the past. The clean-up of polluted soils and waters is very costly, and for many pollutants no feasible technologies are yet available. Plants possess highly efficient systems that acquire and concentrate nutrients as well as numerous metabolic activities, all of which are ultimately powered by photosynthesis. The term phytoremediation has been coined for the concept that plants could be used for low-cost environmental clean-up and this has attracted considerable attention in the past decade [4–6]. During the 1980s, the US Government initiated a large program for the develop- ment of environmental clean-up technologies (The Comprehensive Environmental Response, Compensa- tion, and Liability Act or Superfund), which has acceler- ated the growth of a new productive research field worldwide. As a result, researchers have come to learn that the development of phytoremediation technologies requires a thorough understanding of the underlying processes at the genetic, molecular, biochemical, physio- logical and agronomic levels. This is a review of recent developments in basic and applied research relevant for the plant-based clean-up of soils contaminated with trace metals and metalloids. Available phytoremediation approaches and technologies There are two distinct strategies in soil phytoremediation, phytostabilization and phytoextraction [5]. The former is used to provide a cover of vegetation for a moderately to heavily contaminated site, thus preventing wind and water erosion. Plants suitable for phytostabilization develop an extensive root system, provide good soil cover, possess tolerance to the contaminant metals, and ideally immobilize the contaminants in the rhizosphere. Phytost- abilization is often performed using species from plant communities occurring on local contaminated sites. The most effective but also technically the most difficult phytoremediation strategy is phytoextraction. It involves the cultivation of tolerant plants that concentrate soil contaminants in their above-ground tissues. At the end poplar [15,16]. The MerB protein is an organomercurial lyase that catalyzes the removal of Hg(II) from organic mercury compounds (e.g. methylmercury; Figure 1a). The MerA protein, a mercuric ion reductase, reduces Hg(II) to the volatile elemental form Hg(0) using NADPH as an electron donor. Transgenic plants trans- formed with both merA and merB were remarkably toler- ant to organic mercury compounds and Hg(II). With respect to soil clean-up, the approach is still limited by a generally very low solubility of mercurial compounds in the soil solution. The reaction catalyzed by MerB limited the performance of the plants transformed with merA and merB. Enhanced specific in planta MerB activities were achieved by targeting the MerB protein to the cell wall or to the endoplasmatic reticulum, where the apolar orga- 134 Plant As(SR)3 Vacuolar sequestration by endogenous transport systems ? -CH3(from SMM or AdoMet) MeSeCys Exclusion from protein synthesis MeSe-SeMe ↑ Volatilization? Glu (E) + Cys (C) → γ-Glu-Cys (γ-EC) SeCys SMTA(c) Current Opinion in Biotechnology Chemical reactions in transgenic phytoremediation. (a) The detoxification and volatilization of organomercurials. (b) Arsenate detoxification and immobilization. (c) Selenite detoxification. Gene names are explained in the text. AdoMet, S-adenosylmethionine; GSH, glutathione (reduced); GS-SG, oxidized glutathione; Me, methyl; SMM, S-methylmethionine. contain substantially higher concentrations of the con- taminant than the polluted soil. To achieve this, several bottleneck processes limiting trace element accumulation in plants have to be resolved, including the mobilization of poorly available contaminant trace elements in the soil, root uptake, symplastic mobility and xylem loading, as well as detoxification and storage inside the shoot [7��]. Metal hyperaccumulator plants are naturally capable of accumulating trace elements, primarily Ni, Zn, Cd, As or Se, in their above-ground tissues, without developing any toxicity symptoms [8]. The concentrations of these ele- ments in dry leaf biomass are usually up to 100-fold higher than the concentrations in the soil [4]. Characteristically, the shoot:root ratio of concentrations of the hyperaccu- mulated trace element is above unity [9,10��,11��]. Although metal hyperaccumulator plants therefore appear to have ideal properties for phytoextraction, most of these plants produce little biomass and are thus pri- marily used as model organisms for research purposes. The biomass production of a few hyperaccumulator plants has been judged sufficient for phytomining (the use of plants to extract and concentrate inorganic sub- stances of economic value from soils) [12] or phytoreme- diation; for example, the brake fern Pteris vittata accumulated up to 7500 mg g�1 As on a contaminated site [13], without showing toxicity symptoms. One fern cultivar is available commercially for As phytoextraction (http://www.edenspace.com/index.html), and promising field trials have been conducted [14�]. Chelator-assisted phytoremediation is also available commercially. This approach is based on the application of chelators such as EDTA (ethylenediamine tetraacetate) to solubilize poorly available metals (e.g. lead) in the soil, followed by the largely passive accumulation of metal complexes in plant shoots with the transpiration stream [5]. Because of their extensive root system, their high biomass and low-input cultivation, trees are attractive phytoreme- diators. Metal accumulation is generally poor, however, especially in the wood. Recent genome sequencing, the development of genomics tools, and the ease of genetic transformation of poplar might open up new avenues for the use of trees in phytoremediation [7��]. Breakthroughs in phytoremediation: novel transgenic approaches A variant of phytoextraction is phytovolatilization, whereby the contaminant is not primarily accumulated in above-ground tissues, but is instead transformed by the plant into a volatile compound that is released into the atmosphere. In some groundbreaking work, the detox- ification of highly toxic organomercurial compounds and subsequent volatilization of elemental mercury were successfully engineered in plants. For this purpose, mod- ified bacterial merA and merB genes were introduced into several plant species including Arabidopsis, tobacco and Current Opinion in Biotechnology 2005, 16:133–141 Figure 1 R-CH2-Hg+ + H+ Hg(II) + NADPH AsO43– + 2 GSH → GS-SG + H2O + AsO33– MerB MerA ArsC γ-ECS (a) (b) R-CH3 + Hg(II) → → Hg(0)↑ + NADP+ + H+ Release into the air Shoot: nomercurials are believed to accumulate [17]. Targeting the MerB enzyme to the cell wall is an example for the transfer of a phytoremediation-active compound to the www.sciencedirect.com extracytoplasmic surface of plant cells. Similar ap- proaches may help to solve the problem of low uptake rates of contaminants by plant cells, which still limits all emerging phytoextraction technologies. In order for transgenic phytoremediation to become more widely accepted, the development and implementation of bio- logical encapsulation strategies will be of high value. Biological encapsulation is a term to describe procedures that dramatically decrease the probability of the spread of a transgene from a genetically modified crop to natural plant populations, for example by introducing the trans- gene into the chloroplast genome instead of the nuclear genome [18]. plants on agarose-based media (Table 1). The efficiency of this approach in As phytoextraction remains to be tested on contaminated soils. The expression of ArsC in plants also increases Cd tolerance and accumulation [21��]. Alternatively, strategies to engineer plants for phytoex- traction can be designed based on naturally selected metal hyperaccumulation mechanisms. Transgenic A. thaliana plants expressing a selenocysteine methyltrans- ferase (SMTA) isolated from the Se hyperaccumulator Astragalus bisulcatus accumulated methylselenocysteine and contained up to eightfold higher Se concentrations than wild-type plants, when grown on a soil supplemen- Phytoremediation Kra¨mer 135 xp cea d i A second example further illustrates how well-conceived plant engineering strategies have been designed based on microbial detoxification pathways. In plants, As is taken up as arsenate (AsO4 3�) by phosphate uptake systems [19]. Arabidopsis plants were generated that overexpress an Escherichia coli arsenate reductase ArsC (Figure 1b), which reduces arsenate to arsenite (AsO3 3�) using glu- tathione as the electron donor [20��]. Because arsenite possesses a high affinity for thiol groups and is thus likely to be bound and immobilized inside the cells where it is formed, the arsC gene was introduced downstream of the soybean SRS1 (small subunit of Rubisco 1) promoter, which confers shoot-specific, light-induced expression. The rationale behind this was to keep As immobilization in the root to a minimum in order not to interfere with the translocation of As to the shoot. The resulting transgenic plants were hypersensitive to arsenate, which was attrib- uted to the depletion of the glutathione pool and to the high affinity of arsenite for binding to protein thiol groups. Arabidopsis thaliana plants transformed with arsC and an additional second transgene, which encoded the E. coli g-glutamylcysteine synthetase (g-ECS) expressed under a constitutive actin (ACT2) promoter, were more tolerant to arsenate than the wild-type or single g-ECS transfor- mants. Double-transformant lines accumulated up to 3.4-fold higher shoot As concentrations than wild-type Table 1 Summary of genes introduced into plants and the effects of their e Gene(s) Product/function Source Target arsC Arsenate reductase E. coli Tobacco arsC and g-ECS Arsenate reductase and g-EC synthetase E. coli Arabidopsis E. coli SMTA Selenocysteine Methyltransferase A. bisulcatus Arabidopsis SMTA Selenocysteine methyltransferase A. bisulcatus Brassica jun YCF1 Vacuolar sequestration of GSH–conjugates S. cerevisiae Arabidopsis HMA4 Cellular metal efflux A. thaIiana Arabidopsis aThe ‘maximum effect’ is the maximum concentration increase observe transgene. For a summary of earlier data see [6]. bValue likely to refer to c dimethyl diselenide (MeSe-SeMe). dData were from a single transformant www.sciencedirect.com ted with selenite (SeO3 2�; see Figure 1c and Table 1) [22�]. However, the transgene did not confer tolerance to or enhanced accumulation of Se when the metalloid was provided in the predominant chemical form present in soils, selenate (SeO4 2�). The next important step will be to enhance the rate-limiting conversion of selenate to selenite by the plants. In Brassica juncea seedlings expres- sing the same SMTA protein, another research group observed slight selenate tolerance and an approximately fivefold increase in Se accumulation when plants were exposed to selenate [23�]. They report slightly enhanced dimethyl diselenide volatilization from mature transgenic B. juncea plants exposed to selenite or selenate. Novel insights from basic research In the past few years, substantial progress has been made in elucidating the mechanistic basis of the homeostasis and detoxification of metals and metalloids in plants. Insights into the mechanisms of naturally selected plant trace element tolerance and hyperaccumulation have also been gained. On the basis of this work, improved engi- neering strategies can now be devised. For example, the current transgenic approach in As phytoremediation (described above) could be combined with the expression of phosphate transporters, which appear to be the major uptake pathway for the chemically similar arsenate anions ression on trace element accumulation in shoots. Expression Maximum effecta Root medium Shoot 1.4-fold Cd Hydroponic Shoot and constitutive Threefold As from AsO4 3� Agar Constitutive Eightfold Se from SeO3 2� Amended soil Constitutive bFivefold from SeO4 2� cVolatilization Agar Sand Constitutive 1.4-fold Pb, 1.5-fold Cd Gravel/hydroponic Constitutive dTwofold Zn, 1.4-fold Cd Hydroponic n shoot dry biomass, relative to control plants not expressing the oncentration in whole seedlings. cThe volatilized compound is line only. Current Opinion in Biotechnology 2005, 16:133–141 136 Plant in the roots. There may even be the possibility to isolate mutant or variant phosphate uptake systems with an enhanced affinity for arsenate. The root systems of the As hyperaccumulating fern P. vittata possess a higher affinity for arsenate uptake than those of a related non- accumulator fern species [24�]. A suppression of endo- genous arsenate reduction in roots may serve to enhance root-to-shoot translocation of As [20��,25], and the over- expression of a glutathione–conjugate pump in the leaves could increase the capacity for detoxification of As(III)– glutathione complexes in the vacuole. Finally, phytoche- latins [26] — metal-chelating molecules of the general formula (g-GluCys)nGly (where n = 2 to 11) synthesized by the ubiquitous plant enzyme phytochelatin synthase [27–29] — are known to contribute to As detoxification in As-sensitive [30], As-tolerant [31], and As hyperaccumu- lator plants [32�,33�]. It is interesting to note that the ability to synthesize phytochelatins reduces the extent of cellular As tolerance conferred by expression of the cellular arsenite efflux transporter ScAcr3p in Schizosac- charomyces pombe [34�]. Genomics approaches are increasingly being employed in phytoremediation-related research. Recently, powerful ‘ionomics’ screens have been initiated [35��,36]. These involve unbiased multi-element profiling in A. thaliana mutant populations to identify mutants with altered ele- mental composition of rosette leaves. These and similar screens will serve to identify novel genes with a key role in metal accumulation. Using A. thaliana oligonucl- eotide microarrays, cross-species transcript profiling was employed to compare A. thaliana and a closely related Zn- and Cd-tolerant Zn hyperaccumulating accession of Ara- bidopsis halleri [10��,11��]. This study confirmed on a genome-wide scale what had been observed earlier for single genes in several different hyperaccumulator spe- cies [37–39,40��,41�]: several metal homeostasis genes are constitutively expressed at very high levels in metal hyperaccumulators, when compared with closely related non-accumulators. In A. halleri, these include genes encoding several membrane transporter proteins of the ZRT-IRT-related protein (ZIP) family (zinc-regulated transporter, iron-regulated transporter) [42], which are likely to mediate zinc influx into the cytoplasm, and two isoforms of the enzyme nicotianamine synthase. These genes are expressed at low levels or only upregu- lated under conditions of zinc deficiency in A. thaliana. Other genes found to be constitutively expressed at high levels in the hyperaccumulator species A. halleri encode membrane transport proteins of the HMA (heavy metal P-type ATPase) family of P1B-type metal ATPases, which are potentially involved in metal export into the apoplast for metal detoxification or for root-to-shoot metal trans- location in the xylem. Finally, the transcript analyses implicated a metal tolerance protein 1 (MTP1)-like pro- tein of the so-called cation diffusion facilitator family, which contributes to the sequestration of Zn ions primar- Current Opinion in Biotechnology 2005, 16:133–141 ily in leaf vacuoles (see below). Global transcript analyses of metal hyperaccumulation and associated tolerance in A. halleri are thus consistent with an involvement of several major genes, as supported by preliminary genetic analyses [43�,44,45�]. Collating the available data shows that in several cases homologues of the same A. thaliana gene are overexpressed in various hyperaccumulator species: A. halleri (hyperaccumulating primarily Zn), Thlaspi caeru- lescens (Zn), a different accession of T. caerulescens (Cd, Zn), and Thlaspi goesingense (Ni). Together with site-directed and random mutagenesis approaches, the comparison of amino acid sequences of metal transporters from several hyperaccumulator species might be a starting point in the identification of determinants of differential metal specificities [46]. For example, a sequence compar- ison of the IRT1 protein from A. thaliana and related proteins from T. caerulescens accessions differing in their Cd accumulation might help to identify regions and amino acids that specify Cd transport or exclusion [47�]. Recent work (see also above) suggests a role for nicotia- namine as a chelator conferring cellular tolerance to Ni [48�] and Zn [10��,11��] in a Zn hyperaccumulating accession of T. caerulescens and in A. halleri, res