The major challenge facing society in the twenty-first century is to feed and provide for increasing numbers of people while protecting human health and the environment. To accomplish this we must combine traditional technologies with modern technologies. Contamination of soil and water by industrial effluents and sewage waste is one of the major problems faced by the modern world. The intensive use of potentially toxic compounds by industry and past failures to properly dispose of hazardous material particularly toxic metals now necessitate new methods for the remediation of polluted soil and water. Research efforts are currently being directed at the development of being less invasive, more economical plant-based phytoremediation technology in removing toxic pollutants particularly toxic metals.
Plants have a remarkable ability to extract and concentrate elements and compounds from air, water, and soil. They spend most of their lives as solar-driven pumping stations and chemical factories. Recently, attempts have been made to harness this ability for purposes of environmental remediation. The term phytoremediation has been introduced to describe this process. Phytoremediation is the use of plants to remove pollutants from the environment or to render them harmless. This is being developed as a technology for remediating volatile and nonvolatile organic and toxic metal pollution. However, removal of toxic metals from soils is an area in which phytoremediation may have a particular impact because of the lack of alternative technologies that are affordable and effective.
Plants that hyperaccumumulate toxic metals are rare. Such hyperaccumulators are taxonomically widespread throughout the plant kingdom. More than 350 species of plant are known to accumulate metal such as nickel, zinc, copper, cadmium, selenium or manganese in high levels. For example, naturally occurring hyperaccumulating plants like Thlaspi caerulescens, Serbetia accuminata, Alyssum and Astragolum species which acquire in their tissues high levels of metals such as cadmium, zinc, nickel, have been shown to sequester more than 1% of their dry mass of heavy metals from contaminated soil.
Over the past 10 years, many crop and related weed species have been screened for metal uptake, translocation and tolerance. Much effort has been focussed on the Brassica family to which many hyperaccumulators species belong. However, the potential for application of hyperaccumulators in phytoremediation is limited by several factors such as slow growing, generate insufficient biomass for practical large-scale application, and demonstrate affinity for only one or two toxic elements. An exception is the Indian mustard (Brassica juncea L).
Use of biotechnology to improve Phytoremediation
The lack of affordable effective approaches to remediate soil and water contaminated with toxic metals has created a major need for development of novel approaches including biotechnology. The technology to genetically engineer plants has developed rapidly over the past 15 years. Now it is possible to transfer genes from any organism on our planet to a wide variety of plants. Techniques for transferring foreign genes to individual plant cells, and the subsequent regeneration of these cells into complete plants, have been reported in most major crop plants. Such genetic engineering techniques are enabling scientists to pursue exciting novel strategies. Biotechnology is not a cure-all, but does provide tools with which we can address present-day problems while expanding our comprehension of natural process to prevent future environmental decay.
As we know that traditional plant breeding can only use available genetic diversity within a species to combine the characteristics needed for successful phytoremediation. Therefore, biotechnology approaches to develop transgenic crops for better phytoremediation of toxic metals have been examined. Many genes have been identified and cloned. Now it is possible to express these genes in non-accumulating plants in order to turn them into metal-accumulating plants which can be further used in phytoremediation. Before the genes could be moved from small, slow growing metal hyperaccumulating plants into the fast growing, large non-accumulating plants, and one should consider and understand the molecular aspects of metal hyperaccumulation in the tissues of plant.
Genes for hyperaccumulation
Several steps are required to acquire, transport, and sequester hyperaccumulated metals in the plants. Firstly, metals must cross from the soil solution across root cell plasma membrane into the roots. Secondly, the metals must be loaded into the xylem for translocation to the shoots, in the transpiration streams. Thirdly, metals arriving in the shoot must cross the leaf cell plasma membrane and enter the leaf cell. Finally, the metals are detoxify inside the leaf cell by compartmentalization in the vacuole or transformation into less toxic forms.
The transition of metals from the abiotic to the biotic world takes place at the root/soil interface. For root uptake to occur metals must be released from the solid phase of soil and there should be an existence of efficient system for the mobilization of metals in the rhizosphere of the plant. Hence, bioavailability of toxic metals in soils is critical for effective phytoremediation.
Phytosiderophores are a class of metal-chelating compounds produced by the roots of graminaceaous species to enhance the solubility and uptake of certain cationic micronutrients in the rhizosphere. These compounds have a high binding affinity for iron (III) and the intact phytosiderophore-iron (III) complex is directly transported across the root plasma membrane by specialized membrane transport proteins. Nicotianamine is the main precursor compound for the biosynthesis of the phytosiderophores 2’-deoxymugineic acid and avenic acid. All three of these compounds have the ability to chelate iron (III) and are involved in the mobilization of iron for uptake into the plants. Several related genes, including NAS1, have been cloned from barley that encode nicotianamine synthase, an enzyme which catalyase the synthesis of nicotinanamine from S-adenosylmethionine. The next gene in the phytosiderophore synthesis pathway, nicotianamine aminotransferase (NAAT), has also been cloned from barley.
Metallothioneins (MTs) are a group of small (around 60 amino acids) cysteine-rich eukaryotic proteins that bind heavy metals. Four mouse MTs have been cloned and thoroughly characterized at the molecular level. They consist of two domains and are able to bind a total of seven divalent metal ions. Thus, the overexpression of such proteins in transgenic plants is a promising strategy for the production of plants with superior heavy metals phytoextration capacity.
Regardless of their soil solubility, metals must cross the root cell plasma membrane to gain entry into the plant. There have been several physiological studies on bacteria, yeast, and plants which have revealed the existence of specialized system for the transport of metals across the plasma membrane. The Saccharomyces cervisiae genes ZRT1 and ZRT2 encode proteins for plasma membrane zinc transport. Homologues of these plasma membrane transporters have been cloned from Arabidopsis thaliana. This gene has been designated IRT1 for iron regulated transporter.
A related family of IRT1- like genes has recently been cloned from Arabidopsis thaliana. This family includes ZIP1, ZIP2, ZIP3 and ZIP4. These genes along with IRT1, ZRT-1 and ZRT-2, have been placed in the metal transporter gene family designated ZIP for ZRT, IRT- like proteins. Another family of plasma membrane metal transporters is the Nramp (Natural resistance associated macrophage protein) family. Proteins of the Nramp family contain ten membrane-spanning domains in contrast to the eight membrane-spanning domains of proteins in the ZIP family. Nramp genes were originally found in mammals and yeast, but several representatives have now been cloned from plants.
The next step that an assimilated metal undergoes in a hyperaccumulator plant is loading into the xylem and translocation to the shoot. Low molecular weight metal chelate molecules such as amino acids (e.g. histidine) or organic acids may mediate xylem loading for translocation to the shoots, in the transpiration stream. In general, the shoot to root metal concentration ratio is greater than one in hyperaccumulator plants. This observation implies that hyperaccumulator plants have a more efficient root to shoot translocation system for metals than non-accumulators.
Finally, the plant must be able to tolerate high level of element in the cells. Thus, hypertolerance is the key property which makes a hyperaccumulation possible. It has been found that metals appear to be concentrated in the sub-epidermal and epidermal cells of hyperaccumulator. Hypertolerance is believed to result from vacuolar compartmentalization and chelation. There are some genes that have been identified to be involved in the tonoplast transport of hyperaccumulated metals. At present, only thirteen genes have been identified as being homologues of, or potential candidate genes for transport proteins that may be involved in the intracellular transport of hyperaccumulated metals. Four are from mammals (ZnT1-4), two are from yeast (CoT1 and ZRC1) , four are from A. thaliana (ZAT and AtMTP2-4), and three are from T. goesingense (MTP1, MTP2 and MTP3). Thus, the ZnT, COT1, ZRC1, ZAT and the MTP gene families are promising candidates for vacuolar metal transporters.
Several other genes have now been characterized which encode proteins involved in vacuolar membrane transport of cadmium chelated by either phytochelatin or glutathione. Glutathione (g-glu-cys-gly) plays several important roles in the defence of plants against environmental stresses, and is the precursor for phytochelatins. Glutathione is synthesized in two enzymatic reaction, catalysed by glutamylcysteine synthase (ECS) and glutathione synthase (GS), respectively. Phytochelatins are a class of compounds that has been shown to be involved in transporting cadmium across the tonoplast membrane into vacuoles. These small molecules weight compounds are synthesized by plants in response to a number of metals such as cadmium, lead, zinc, nickel, and mercury. Phytochelatins are enzymatically synthesized from the precursors g-glutamylcysteine and glutathione by PC synthase. Recently, genes for all the biosynthetic steps required to convert cystein, glutamate and glycine into phytochelatins have been cloned.
Thus, a fundamental understanding of the biochemical processes involved in plant metal uptake, translocation and hyperaccumulation in normal and metals hyperaccumulators, regulatory control of these activities, and the use of tissue-specific promoters offers great promise that use of molecular biology tools can give scientist the ability to develop effective and economic phytoremediation transgenic plants for toxic metals. So, a long term effort should be directed towards developing a “molecular tool box” composed of genes valuable for phytoremediation.
Metal hyperaccumulation is an extraordinary property of a few specialized plant species. Therefore, the genetic material of these specialized plants can be exploited to enhance the nutritional properties of crop plants ideally suited to phytoremediation. As we know that phytoremedation is a nascent technology that seeks to exploit the metabolic capabilities and growth habits of plants, this will require a multidisciplinary approach as diverse as plant biology, agronomy, microbiology, biochemistry, agricultural engineering, soil science and genetic engineering, before we can take full advantage of the genetic potential of these plants.