Proteomics in Phytoremediation Research

At least two approaches have been used to characterize and apply knowledge of plant enzymes in the sustainable phytodegradation of organic pollutants. These include the following types of investigations.

1. Natural reactions, usually very fast, dominate transformations, are traced to plant and microbial enzymes.

2. Roles for well-characterized enzymes are developed.

Microarrays of common contaminants, secondary metabolites, or typical enzymatic activities (which could be used simultaneously to develop pharmaceuticals and nutraceuticals) do not seem to have been applied to explore untapped plant enzymatic activities.

All approaches to applying enzymatic activity to phyto-remediation involve some element of trial and error, and are thus similar to the development ofnew medicines. The trial-and-error testing of plant and insect samples from diverse but unusual settings like jungles, deserts, or evolutionarily isolated environments to discover high metabolic and other activities is the same as reacting representative compounds with environmental samples to discover natural transformations of contaminants in different media. In most cases, classical techniques in biochemistry are used or have been used to identify and purify the enzymes and other proteins responsible for the activity. A pathway analysis and characterization of the contaminant fate is equally important to efficiently guide the characterization of the enzymes reacting with and controlling pollutant fate and transport. Early insight into potentially more toxic metabolites must be followed with interim toxicity tests, including phytotoxicity tests to select the best plants to apply.

Natural reactions have been explored in two complementary ways. One focused initially on enzyme isolation and characterization, the other on inferring enzymatic activity from contaminant transformation pathway analysis. Both were necessary to comprehensively define and apply the reactions involved.

The most useful approach to developing new phytodegradation applications to date is to start with observations of a fast natural reaction of a compound of interest and isolate and characterize the active components. In three of three isolations from reducing sediments, stable plant enzymes in reducing sediments have proven to be responsible for the fast reactions discovered, not microbial enzymes as expected. Starting with fast natural reactions or unusual accumulations of metals and inorganic compounds usually ensures that an inexpensive in situ process is possible, and some suboptimal, black box applications are always possible before full characterization of the natural processes has occurred.

Starting with a known enzyme like peroxidases (EC 1.11.17) or phosphatases (EC 3.1.3) and finding roles in phytoremediation is much less successful so far. Potential applications are generally highly controlled ex situ unit processes that provide vital cofactors and control of toxic intermediates, if applications can be developed at all. High degrees of control are usually unsustainably expensive and energy intensive, but normally more reliable in meeting cleanup standards.

The proteomics of phytodegradation seems to be more broadly understood than that for metals accumulation by plants. There are at least three bodies of knowledge that contribute to the breadth of information on plant proteo-mics used in phytodegradation practice today. First, the extensive investigation of plant proteomics for the development of pesticides and herbicides used in agriculture provides invaluable insights into not only plant tolerance, but more importantly metabolism, detoxification, and transformation of different classes of organic compounds. Much of this experience is readily extrapolated to waste management using plants because pesticides and herbicides are important contaminants at some sites, especially worldwide. More importantly, pesticide development has been carefully organized in terms of plant effects and the structure and activity of the synthesized pesticides, which is easily extrapolated to similar contaminants. The most significant drawback is that much of the research is proprietary and has never been published, especially for candidate compounds not developed into pesticides.

Second, quite a few enzymes are common to microbes and plants and the field of bioremediation has pioneered exploration of the proteomics and more recently the genetics of some important enzymatic processes. The pioneering pathway analyses for bioremediation are a vital tool used to accelerate the acceptance and use of phytoremediation.

Third, the vital insight that plants are green livers means that the more extensive medical and veterinary research on the proteomics, genetics, and genomics of cell biology could be translated in the development of phytoremedia-tion using the seminal differences between plant and animal cells as a basis for extrapolation. Unfortunately, medical research seems to have less strict process endpoint requirements in some cases and the rates of transformations are not always defined well enough for immediate extrapolations to phytoremediation applications.

The impact of other research on plant biochemistry does not seem to have been as important, but seminal research on allelochemicals and natural pesticides and synthesis and transformation of plant metabolites should prove vital in the future. Knowledge of the approximate 200 000 plant metabolites should be vital in developing new applications and defining the limits of phytoreme-diation. When a plant or any organism produces an allelopathic, poisonous, or other toxic secondary metabolite, there must be a transformation process that prevents these toxic compounds from eventually building up and swamping the planet. An example is plant lignification and delignification that uses peroxidases (EC 1.11.17) both to synthesize and degrade woody tissue. The seminal conclusion is that the existence of a primary or secondary metabolite similar to a contaminant molecule provides a good chance that phytotransformation or ecological processes can be used to degrade and control the waste sustainably, given enough time and land area. The approximate 200 000 plant secondary metabolites alone provide up to 200 000 potential metabolic pathways for degradation. However, a number of these pathways will share common transformations, the number of which may not have been estimated yet.

The enzymatic processes involved in metals accumulation by plants may not be as broadly explored as for organic metabolism and detoxification, but those investigations that have been undertaken for cadmium, lead, mercury, nickel, zinc, and a few other elements do delve deeply into the genetics and proteomics of these processes. The investigations of the proteomics of metal accumulation are similar to those used to define the role of enzymes in organic transformations and mineralization. Hyperaccumulating plants are the basis of proteomic and genetic characterizations supporting some applications, while specific genes and enzymes have been investigated for other applications of metals 'phytoaccumulation'. For 'phytosorption', much less is known about plant and algae cell wall characteristics and the effects on electrostatic attraction and formation of complexes or the role of polysaccharides, uronic acids, and sulfated polysacchar-ides that can bind heavy metals. Thus the selection of plants and algae species and other design decisions are made by trial-and-error testing at the moment.

'Hyperaccumulation' is arbitrarily defined as occurring when a plant contains more of a metal than 0.01%, 0.1%, or 1% by weight, depending on how frequently the metal occurs in the environment (Table 1). These hyper-accumulating plants are relatively rare and usually consist of slow-growing herbs. Some of these hyperaccumulators seemed to have evolved on metal outcroppings that normally poison plants; some may have evolved to accumulate metals as a natural insecticide or perhaps as an elemental allelopathic defense. Some hyperaccumulators may benefit from 'mutualism'. Mutualism may include (1) mycorrhizal or plant ecosystems that make metals in soils more available and less toxic with chelating ligands or enzymes and (2) accumulation of metals in seed and pollen to select only certain-metal-tolerant animals for the transport of this important genetic material. Commensalism in the form of epiphytism could explain some hyperaccumulation, but some hyperaccumulators cannot be explained functionally or evolutionarily.

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