Advances in development of transgenic plants for remediation of xenobiotic pollutants

Phytoremediation-the use of plants for cleaning up of xenobiotic compounds-has received much attention in the last few years and development of transgenic plants tailored for remediation will further enhance their potential. Although plants have the inherent ability to detoxify some xenobiotic pollutants, they generally lack the catabolic pathway for complete degradation/mineralization of these compounds compared to microorganisms. Hence, transfer of genes involved in xenobiotic degradation from microbes/other eukaryotes to plants will further enhance their potential for remediation of these dangerous groups of compounds. Transgenic plants with enhanced potential for detoxification of xenobiotics such as trichloro ethylene, pentachlorophenol, trinitro toluene, glycerol trinitrate, atrazine, ethylene dibromide, metolachlor and hexahydro-1,3,5-trinitro-1,3,5-triazine are a few successful examples of utilization of transgenic technology. As more genes involved in xenobiotic metabolism in microorganisms/eukaryotes are discovered, it will lead to development of novel transgenic plants with improved potential for degradation of recalcitrant contaminants. Selection of suitable candidate plants, field testing and risk assessment are important considerations to be taken into account while developing transgenic plants for phytoremediation of this group of pollutants. Taking advantage of the advances in biotechnology and 'omic' technologies, development of novel transgenic plants for efficient phytoremediation of xenobiotic pollutants, field testing and commercialization will soon become a reality.     

Secretion and folding of human growth hormone in Escherichia coli

Abstract

Phytoremediation is the use of plants for the treatment of environmental pollution, including chlorinated organics. although conceptually very attractive, removal and biodegradation of chlorinated pollutants by plants is a rather slow and inefficient process resulting in incomplete treatment and potential release of toxic metabolites into the environment. In order to overcome inherent limitations of plant metabolic capabilities, plants have been genetically modified, following a strategy similar to the development of transgenic crops: genes from bacteria, fungi, and mammals involved in the metabolism of organic contaminants, such as cytochrome p-450 and glutathione substrate catabolic genes, natural or engineered, for the simultaneous remediation of a range of pollutants, such as usually found in contaminated sites, e.g., chlorinated solvent, metals, and nitroaromatics. In addition, biodegradation of many xenobiotics are catalyzed by similar, broad-substrate enzymes, such as cytochrome P-450 monoxygenases, glutathione S-transferases, and fungal peroxidases, that can potentially be used for the treatment of multiple pollutants. Moreover, the introduction of multiple transgenes involved in different phases of the metabolism of xenobiotics in plants, i.e., uptake by roots and the different phases of the green liver model, would allow enhancing both the removal and metabolism of several toxic compounds and could therefore help overcome a major limitation inherent to phytoremediation, i.e., the threat that accumulated toxic compounds would volatilize or otherwise contaminate the food chain. An important barrier to the application of transgenic plants for bioremediation in the field is associated with the true or perceived risk of horizontal gene transfer to related wild or cultivated plants. Therefore, it is likely that the next generation of transgenic plants will involve systems preventing such a transfer, for instance by the introduction of transgenes into chloroplastic DNA or the use of conditional lethality genes (Davison, 2005). Since bacteria naturally exchange plasmids via conjugation, endophytes that gain genes involved in pollutant degradation might not be considered ‘genetically modified’ and may be subject to fewer restrictions in usage.     

Transgenic rice plants expressing human p450 genes involved in xenobiotic metabolism for phytoremediation

Phytoremediation is the use of plants to remove xenobiotic compounds from the environment. Plants have the inherent ability to detoxify xenobiotic pollutants, but they are generally poor at degrading them. The introduction of genes involved in xenobiotic degradation is aimed at enhancing plants' potential further. Rice (Oryza sativa) is a good candidate for this purpose and has been transformed with genes encoding cytochrome P450 monooxygenases CYP1A1, CYP2B6, and CYP2C19. The transgenic plants were more tolerant to various herbicides than nontransgenic Nipponbare rice plants, owing to enhanced metabolism by the introduced P450 enzymes. Transgenic plants were able to remove atrazine and metolachlor from soil. Field testing and risk assessment are very important for developing transgenic plants for phytoremediation. Transgenic rice plants should become useful as herbicide-tolerant crops and for phytoremediation of xenobiotic pollutants in future.     

Perspectives of bacterial ACC deaminase in phytoremediation

Phytoremediation of contaminated soil and water environments is regulated and coordinated by the plant root system, yet root growth is often inhibited by pollutant-induced stress. Prolific root growth could maximize rates of hyperaccumulation of inorganic contaminants or rhizodegradation of organic pollutants, and thus accelerate phytoremediation. Accelerated ethylene production in response to stress induced by contaminants is known to inhibit root growth and is considered as a major limitation in improving phytoremediation efficiency. Recent work shows that bacterial 1-aminocyclopropane-1-carboxylate (ACC) deaminase regulates ethylene levels in plants by metabolizing its precursor ACC into alpha-ketobutyric acid and ammonia. Plants inoculated with ACC deaminase bacteria or transgenic plants that express bacterial ACC deaminase genes can regulate their ethylene levels and consequently contribute to a more extensive root system. Such proliferation of roots in contaminated soil can lead to enhanced uptake of heavy metals or rhizodegradation of xenobiotics.     

Phytoremediation--a novel and promising approach for environmental clean-up

Phytoremediation is an eco friendly approach for remediation of contaminated soil and water using plants. Phytoremediation is comprised of two components, one by the root colonizing microbes and the other by plants themselves, which degrade the toxic compounds to further non-toxic metabolites. Various compounds, viz. organic compounds, xenobiotics, pesticides and heavy metals, are among the contaminants that can be effectively remediated by plants. Plant cell cultures, hairy roots and algae have been studied for their ability to degrade a number of contaminants. They exhibit various enzymatic activities for degradation of xenobiotics, viz. dehalogenation, denitrification leading to breakdown of complex compounds to simple and non-toxic products. Plants and algae also have the ability to hyper accumulate various heavy metals by the action of phytochelatins and metallothioneins forming complexes with heavy metals and translocate them into vacuoles. Molecular cloning and expression of heavy metal accumulator genes and xenobiotic degrading enzyme coding genes resulted in enhanced remediation rates, which will be helpful in making the process for large-scale application to remediate vast areas of contaminated soils. A few companies worldwide are also working on this aspect of bioremediation, mainly by transgenic plants to replace expensive physical or chemical remediation techniques. Selection and testing multiple hyperaccumulator plants, protein engineering ofphytochelatin and membrane transporter genes and their expression would enhance the rate of phytoremediation, making this process a successful one for bioremediation of environmental contamination. Recent years have seen major investments in the R&D, which have also resulted in competition of filing patents by several companies for economic gains. The details of science & technology related to phytoremediation have been discussed with a focus on future trends and prospects of global relevance.     

Phytoremediation—A Novel and Promising Approach for Environmental Clean-up

ABSTRACT

Phytoremediation is an eco friendly approach for remediation of contaminated soil and water using plants. Phytoremediation is comprised of two components, one by the root colonizing microbes and the other by plants themselves, which degrade the toxic compounds to further non-toxic metabolites. Various compounds, viz. organic compounds, xenobiotics, pesticides and heavy metals, are among the contaminants that can be effectively remediated by plants. Plant cell cultures, hairy roots and algae have been studied for their ability to degrade a number of contaminants. They exhibit various enzymatic activities for degradation of xenobiotics, viz. dehalogenation, denitrification leading to breakdown of complex compounds to simple and non-toxic products. Plants and algae also have the ability to hyper accumulate various heavy metals by the action of phytochelatins and metallothioneins forming complexes with heavy metals and translocate them into vacuoles. Molecular cloning and expression of heavy metal accumulator genes and xenobiotic degrading enzyme coding genes resulted in enhanced remediation rates, which will be helpful in making the process for large-scale application to remediate vast areas of contaminated soils. A few companies worldwide are also working on this aspect of bioremediation, mainly by transgenic plants to replace expensive physical or chemical remediation techniques. Selection and testing multiple hyperaccumulator plants, protein engineering of phytochelatin and membrane transporter genes and their expression would enhance the rate of phytoremediation, making this process a successful one for bioremediation of environmental contamination. Recent years have seen major investments in the R&D, which have also resulted in competition of filing patents by several companies for economic gains. The details of science & technology related to phytoremediation have been discussed with a focus on future trends and prospects of global relevance.     

Assessing the applicability of phytoremediation of soils with mixed organic and heavy metal contaminants

Soil pollution is a major environmental problem and many contaminated sites are tainted with a mixture of organic and heavy metal contaminants. Compared to other remedial strategies, phytoremediation is a low cost, environmentally-friendly, sustainable means of remediating the contamination. This review first provides an overview of phytoremediation studies where the soil is contaminated with just one type of pollutant (heavy metals or organics) and then critically evaluates the applicability of phytotechnologies for the remediation of contaminated sites where the soil is polluted by a mixture of organic and heavy metal contaminants. In most of the earlier research studies, mixed contamination was held to be detrimental to plant growth, yet there were instances where plant growth was more successful in soil with mixed contamination than in the soil with only individual contaminants. New effective phytoremediation strategies can be designed for remediation of co-contaminated sites using: (a) plants species especially adapted to grow in the contaminated site (hyperacumulators, local plants, transgenic plants); (b) endophytic bacteria to enhance the degradation in the rizhosphere; (c) soil amendments to increase the contaminants bioavailability [chelating agents and (bio)surfactants]; (d) soil fertilization to enhance the plant growth and microbial activity in the soil; and (e) coupling phytoremediation with other remediation technologies such as electrokinetic remediation or enhanced biodegradation in the rhizosphere.     

Microbial expression profiles in the rhizosphere of willows depend on soil contamination

The goal of phytoremediation is to use plants to immobilize, extract or degrade organic and inorganic pollutants. In the case of organic contaminants, plants essentially act indirectly through the stimulation of rhizosphere microorganisms. A detailed understanding of the effect plants have on the activities of rhizosphere microorganisms could help optimize phytoremediation systems and enhance their use. In this study, willows were planted in contaminated and non-contaminated soils in a greenhouse, and the active microbial communities and the expression of functional genes in the rhizosphere and bulk soil were compared. Ion Torrent sequencing of 16S rRNA and Illumina sequencing of mRNA were performed. Genes related to carbon and amino-acid uptake and utilization were upregulated in the willow rhizosphere, providing indirect evidence of the compositional content of the root exudates. Related to this increased nutrient input, several microbial taxa showed a significant increase in activity in the rhizosphere. The extent of the rhizosphere stimulation varied markedly with soil contamination levels. The combined selective pressure of contaminants and rhizosphere resulted in higher expression of genes related to competition (antibiotic resistance and biofilm formation) in the contaminated rhizosphere. Genes related to hydrocarbon degradation were generally more expressed in contaminated soils, but the exact complement of genes induced was different for bulk and rhizosphere soils. Together, these results provide an unprecedented view of microbial gene expression in the plant rhizosphere during phytoremediation.     

Enhancement of metabolizing herbicides in young tubers of transgenic potato plants with the rat CYP1A1 gene

A rat P450 monooxygenase gene (CYP1A1) was introduced into potato plants to enhance the metabolism of the environmental contaminants in subterranean organs. The CYP1A1 gene was kept under the control of the potato patatin promoter to enhance tuber-specific expression. A total of 106 transgenic plants (PAT1A1 plants) were obtained following selection by a resistance test to kanamycin and PCR analysis. PAT1A1 plants treated with 10% exogenous sucrose showed a higher activity of monooxgenase in the leaves than the non-transgenic plants. This indicated that the activity enhanced by 10% sucrose was due to the patatin promoter containing the sucrose-inducted elements. One representative transgenic plant, Ag2197, was selected on the basis of monooxgenase activity in the leaves and Western blot analysis. Ag2197 was found to accumulate a large amount of CYP1A1 mRNA and protein in the developing tuber but not in the mature tuber. The residual herbicides, atrazine and chlortoluron, were analyzed in the micro-tubers of Ag2197 and non-transgenic plants. The amount of residual herbicides in Ag2197 was much lower than that in the non-transgenic plant, indicating that the transgenic plant metabolized the herbicides to a detoxified form. The transgenic plants produced in this study might be useful for the phytoremediation of chemical pollution in the soil.     

Phytoremediation of the organic Xenobiotic simazine by p450-1a2 transgenic Arabidopsis thaliana plants

The potential use of human P450-transgenic plants for phytoremediation of pesticide contaminated soils was tested in laboratory and greenhouse experiments. The transgenic P450 CYP1A2 gene Arabidopsis thaliana plants metabolize number of herbicides, insecticides and industrial chemicals. The P450 isozymes CYP1A2 expressed in A. thaliana were examined regarding the herbicide simazine (SIM). Transgenic A. thaliana plants expressing CYP1A2 gene showed significant resistance to SIM supplemented either in plant growth medium or sprayed on foliar parts. The results showed that SIM produces harmful effect on both rosette diameter and primary root length of the wild type (WT) plants. In transgenic A. thaliana lines, the rosette diameter and primary root length were not affected by SIM concentrations used in this experiment. The results indicate that CYP1A2 can be used as a selectable marker for plant transformation, allowing efficient selection of transgenic lines in growth medium and/or in soil-grown plants. The transgenic A. thaliana plants exhibited a healthy growth using doses of up to 250 μmol SIM treatments, while the non-transgenic A. thaliana plants were severely damaged with doses above 50 μmol SIM treatments. The transgenic A. thaliana plants can be used as phytoremediator of environmental SIM contaminants.     

Phytoremediation of organochlorine pesticides: Concept,method, and recent developments

Rapid increase in industrialization of world economy in the past century has resulted in significantly high emission of anthropogenic chemicals in the ecosystem. The organochlorine pesticides (OCPs) are a great risk to the global environment and endanger the human health due to their affinity for dispersion, transportation over long distances, and bioaccumulation in the food chain. Phytoremediation is a promising technology that aims to make use of plants and associated bacteria for the treatment of groundwater and soil polluted by these contaminants. Processes known to be involved in phytoremediation of OCPs include phytoaccumulation, rhizoremediation, and phytotransformation. Vegetation has been accounted to considerably amplify OCP elimination from soil, in contrast to non-planted soil, attributable to both, uptake within plant tissues and high microbial degradation of OCP within the root zone. Developing transgenic plants is a promising approach to enhance phytoremediation capabilities. Recent advances in the application of phytoremediation technique for OCPs, including uptake by plants and plant–microbe association in the rhizosphere for the enhanced degradation and mineralization of these pollutants, is presented in this review. Additionally, some attempts to improve this technique using transgenesis and role of certain enzymes are also discussed.     

Phytoremediation: novel approaches to cleaning up polluted soils

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 populations for a genetic analysis of naturally selected metal hyperaccumulation in plants, and from comprehensive ionomics data--multi-element concentration profiles from a large number of Arabidopsis mutants.     

植物中多氯联苯的来源、分布及代谢研究进展

研究植物中多氯联苯（PCBs）的来源、分布和代谢特点,可以更好地发挥植物在PCBs环境监测中的被动采样平台作用,丰富植物修复PCBs污染的基础理论。对植物中PCBs的来源途径方面的已有研究进行了总结,阐述了植物吸收PCBs的机制及影响因素,论述了植物不同部位中PCBs的分配特点,概括了PCBs的代谢机理和应用的研究现状,最后指出了目前存在的问题和未来的研究方向。     

Can tobacco have a potentially beneficial effect to our health?

With urgent pressure to clean up the contaminated environment, new approaches are needed. Phyto- and rhizoremediation using plants and related bacteria is a promising approach, but has its inborn limitations. To overcome the slow performance of the process, transgenic plants have been prepared specifically tailored for phytoremediation purposes. Our projects addressed a group of widespread synthetic organic xenobiotics, polychlorinated biphenyls (PCBs), and heavy metals as representatives of inorganic contaminants. Beside basic research studies in the field of phyto/rhizoremediation of the mentioned toxicants we focused on genetically modified plants as a highly promising tool for these purposes. We tried to prepare tobacco plants expressing the bacterial enzyme responsible for cleaving PCBs, coded by the gene bphC from the bacterial biphenyl operon. The expression of bphC product in fusion with the green fluorescent protein is described together with evaluation of the twice increased resistance of transgenic seeds towards PCBs. The other model is addressing improvement of cadmium accumulation by preparing plants bearing fused transgenes of metal binding protein (yeast metallothionein) with an introduced additional metal binding domain--polyhistidine anchor with high affinity to metals. The genetically modified plants exhibit 190% Cd accumulation of the control in harvestable parts, higher resistance and lower Cd content in roots. The performance of the plants in real contaminated soil is also evaluated.     

植物修复多氯联苯研究进展

综述了植物修复持久性有机污染物多氯联苯（PCBs）的研究进展,重点阐述了植物对PCBs的去除作用和机理,植物在从环境中去除PCBs的过程中,不仅仅是作为微生物降解的支持者,而且还作为积极的参与者对PCBs进行代谢：一方面植物通过根系从环境中吸收和积累PCBs,并将吸收的PCBs转化为非毒性的代谢产物累积于植物组织中;另一方面植物释放促进PCBs降解的酶直接降解PCBs,或释放根系分泌物,增加根际微生物的数量,提高其活性间接降解PCBs.文中对植物修复PCBs的影响因素如植物组织培养的类型、生物量、PCBs的初始浓度以及PCBs的类型、理化性质等进行了讨论.     

Expression of bacterial biphenyl-chlorobiphenyl dioxygenase genes in tobacco plants

Optimized plant-microbe bioremediation processes in which the plant initiates the metabolism of xenobiotics and releases the metabolites in the rhizosphere to be further degraded by the rhizobacteria is a promising alternative to restore contaminated sites in situ. However, such processes require that plants produce the metabolites that bacteria can readily oxidize. The biphenyl dioxygenase is the first enzyme of the bacterial catabolic pathway involved in the degradation of polychlorinated biphenyls. This enzyme consists of three components: the two sub-unit oxygenase (BphAE) containing a Rieske-type iron-sulfur cluster and a mononuclear iron center, the Rieske-type ferredoxin (BphF), and the FAD-containing ferredoxin reductase (BphG). In this work, based on analyses with Nicotiana benthamiana plants transiently expressing the biphenyl dioxygenase genes from Burkholderia xenovorans LB400 and transgenic Nicotiana tabacum plants transformed with each of these four genes, we have shown that each of the three biphenyl dioxygenase components can be produced individually as active protein in tobacco plants. Therefore, when BphAE, BphF, and BphG purified from plant were used to catalyze the oxygenation of 4-chlorobiphenyl, detectable amounts of 2,3-dihydro-2, 3-dihydroxy-4'-chlorobiphenyl were produced. This suggests that creating transgenic plants expressing simultaneously all four genes required to produce active biphenyl dioxygenase is feasible.     

Plant growth promoting rhizobacteria and endophytes accelerate phytoremediation of metalliferous soils

Technogenic activities (industrial—plastic, textiles, microelectronics, wood preservatives; mining—mine refuse, tailings, smelting; agrochemicals—chemical fertilizers, farm yard manure, pesticides; aerosols—pyrometallurgical and automobile exhausts; biosolids—sewage sludge, domestic waste; fly ash—coal combustion products) are the primary sources of heavy metal contamination and pollution in the environment in addition to geogenic sources. During the last two decades, bioremediation has emerged as a potential tool to clean up the metal-contaminated/polluted environment. Exclusively derived processes by plants alone (phytoremediation) are time-consuming. Further, high levels of pollutants pose toxicity to the remediating plants. This situation could be ameliorated and accelerated by exploring the partnership of plant-microbe, which would improve the plant growth by facilitating the sequestration of toxic heavy metals. Plants can bioconcentrate (phytoextraction) as well as bioimmobilize or inactivate (phytostabilization) toxic heavy metals through in situ rhizospheric processes. The mobility and bioavailability of heavy metal in the soil, particularly at the rhizosphere where root uptake or exclusion takes place, are critical factors that affect phytoextraction and phytostabilization. Developing new methods for either enhancing (phytoextraction) or reducing the bioavailability of metal contaminants in the rhizosphere (phytostabilization) as well as improving plant establishment, growth, and health could significantly speed up the process of bioremediation techniques. In this review, we have highlighted the role of plant growth promoting rhizo- and/or endophytic bacteria in accelerating phytoremediation derived benefits in extensive tables and elaborate schematic sketches.     

Biodegradation and biotransformation of explosives

Explosives now contaminate millions of hectares of land in the US alone, with global levels of contamination difficult to fully assess. Understanding the biology behind the metabolism of these toxic compounds by microorganisms and plants is imperative for managing these pollutants in the environment. Towards this aim, recent studies have identified, and are now characterizing, plant genes involved in 2,4,6-trinitrotoluene detoxification and the biochemical pathways of nitramine degradation in microorganisms. A key scientific goal continues to be identification of enzymes capable of degrading 2,4,6-trinitrotoluene and this still remains elusive, although recent reports give insights into the origin of nitrite released during biotransformation of this major contaminant. Promising phytoremediation research using transgenic model plant systems has now been transferred to poplar, a species with field applicability.