Understanding pesticide metabolism in plants is necessary for pesticide development, for safe and efficient use, as well as for developing pesticide phytoremediation strategies for contaminated soil and water. Individual reactions of degradation–detoxification pathways in plants include oxidation, reduction, hydrolysis, and conjugation.
Metabolic pathway diversity depends on the chemical structure of the xenobiotic compound (pesticide), the plant, environmental conditions, metabolic factors, and the regulating expression of these biochemical pathways.
Knowledge of these enzymatic processes, especially concepts related to pesticide mechanism of action, resistance, selectivity, tolerance, and environmental fate, has advanced understanding of pesticide science, and of plant biochemistry and physiology (Van Eerd et al., 2003).
Pesticide Metabolism in Plants
Enzymatic transformation, which is mainly the result of biotic processes mediated by plants and microorganisms, is by far the major route of detoxification. Metabolism of pesticides may involve a three-phase process (Hatzios 1991).
In Phase I metabolism, the initial properties of a parent compound are transformed through oxidation, reduction, or hydrolysis to generally produce a more water-soluble and usually a less toxic product than the parent.
The second phase involves conjugation of a pesticide or pesticide metabolite to a sugar, amino acid, or glutathione, which increases the water solubility and reduces toxicity compared with the parent pesticide.
Generally, Phase II metabolites have little or no phytotoxicity and may be stored in cellular organelles. The third phase involves conversion of Phase II metabolites into secondary conjugates, which are also nontoxic (Hatzios 1991).
In leafy spurge (EuphorbiaesulaL.), examples of Phase III metabolism are the conjugation of the N– glycoside metabolite of picloram with malonate and the formation of a gentibioside from the picloram glucose ester metabolite.
Although there are fundamental similarities and differences between plant and microbial pesticide metabolism, this article will emphasize the enzymatic transformations of a wide variety of pesticides in plants, and present the mechanism, biochemistry, genetics, and regulation of these processes in plants.
Furthermore, the broad aspects of pesticide metabolism in plants as well as the importance of these biochemical pathways for pesticide development and environmental stewardship will be discussed.
Oxygenation is the most frequent first step in the biotransformation of pesticides and other organic xenobiotics, and many of these reactions are mediated by oxidative enzymes. Plants therefore produce a wide range of oxidative enzymes. These include cytochrome P450, peroxidases, polyphenoloxidase, laccase, and tyrosinase, and polyphenol oxidases.
Reactions by Cytochromes P450
The most extensively studied oxidative enzymes in plants are the P450s, which are the most important enzymes in Phase I pesticide metabolism (Barrett, 2000). Cytochrome P450s are hemethiolate proteins that have been characterized in animals, plants, bacteria, and filamentous fungi.
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In plants, P450s produce many secondary metabolites including plant growth regulators, isoprenoids, and alkaloids. Cytochrome P450s are encoded by a superfamily of genes designated as CYP,which have highly conserved residues around the heme portionof the protein (Barrett 2000).
The first plant P450 gene was sequenced in 1990 (Bolwell et al. 1994), and presently, more than 500 P450 plant genes have been described (Barret 2000). P450 genes occur in clusters in the genome.
Regulation and expression of P450s are not well understood in plants mainly because of the very low quantities of P450 enzymes usually present in plant cells, particularly if the plant has not been exposed to physiochemical, physiological, or xenobiotic stress.
Cytochrome P450s often catalyze monooxygenase reactions, usually resulting in hydroxylation. However, there are many other P450-mediated reactions including dehydration, dimerization, deamination, dehydrogenation, heteroatom dealkylation, epoxidation, reduction, and C–C or C5N cleavage.
Among the three classes of P450 found in plants, animals and microorganisms, only class I and class III P450s are found in plants.
Class I P450s are flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) dependent, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) requiring P450s that are usually microsomal membrane-bound proteins in plants and filamentous fungi. Class III P450s are located in plant plastids and do not require auxillary redox partners.
Agrochemicals can influence cytochrome P450 systems by acting as effectors, thereby modifying pesticide metabolism, or by modulating overall metabolism of an organism. These effects can increase or decrease physiological activities, which may affect growth and development.
Direct evidence that xenobiotic metabolism was mediated by P450swas obtained through experimentation with plant microsomal preparations. Using microsomal preparations from several plant species, it was shown that chlortoluron was metabolized to two metabolites by at least two different P450 enzymes.
Since that time, a number of P450- mediated phenylurea-metabolizing genes have been characterized. Mougin et al. (2001) demonstrated that the fungicide fenpropimorph was metabolized to an oxygenated metabolite in wheat seedling microsomal preparations. Increased metabolism occurred when seeds were pretreated with naphthalic anhydride, a chemical safener that enhances cytochrome P450 levels.
Further, oxidation of fenpropimorph in wheat seedling microsomes was inhibited when the preparations were exposed to carbon monoxide, which binds to the heme portion of the P450 molecule instead of oxygen, thereby blocking enzymatic reactions. These authors suggested that fenpropimorph metabolism is P450- mediated.
Other researchers have used microsomes to demonstrate that the mechanism of resistance to several dissimilar herbicide chemistries in blackgrass (Alopecurusmyosuroides) and rigid ryegrass (Loliumrigidum) was based on enhanced P450-mediated metabolism.
Herbicide resistance mediated by P450s mayarise via two scenarios:
(1) mutation of an existing P450, allowing increased binding and metabolism of the herbicide or
(2) increased activity of existing P450s (Barrett 2000).
In the future, researchers will no doubt continue to focus on isolating and characterizing plant P450 genes associated with pesticide metabolism. With a better understanding of P450 genes and their regulation, it may be possible to manipulate the crop plant system to increase herbicide tolerance.
Peroxidases, Phenoloxidases, and Related Oxidoreductases
Other than P450s that catalyze the polymerization of various anilines and phenols (Dec and Bollag2001), there are other peroxidase-mediated pesticide transformations in plants that function similar to P450s.
These include decarboxylation, sulfur oxidation, N-demethylation, ring hydroxylation, and aromatic methyl group oxidations. In plants, peroxidase enzymes often function in Phase III metabolism, e.g., formation of bound residues.
Horseradish (AmorocialapathifoliaGilib.) roots contain large quantities of peroxidase. Horseradish root tissue has been used to remove 2,4-dichlorophenol from water and was more effective in contaminant removal than the purified peroxidase enzyme (Dec and Bollag 2001).
In most instances, polymerization products have reduced toxicity compared with the substrate (Dec and Bollag, 2001).
Hydrolytic enzymes cleave bonds of a substrate by adding H or OH from H2O to each product. There are many hydrolytic enzymes that are capable of metabolizing a variety of substrates, particularly those containing amide, carbamate, or ester functional groups.
These enzymes may be compartmentalized or extracellular, and reactions can occur under aerobic or anaerobic conditions. Like most classes of enzymes, hydrolytic enzymes may have broad substrate specificities, thereby allowing degradation of a variety of pesticides.
Pesticide ester hydrolysis in plants has been extensively studied and reviewed (Hoagland and Zablotowicz 2001). Ester hydrolysis is commonly carried out by esterases and to a much lesser extent by lipases and proteases.
Often, herbicides such as fenoxaprop-ethyl, diclofop-methyl, and 2, 4- DB are esterified to increase absorption and selectivity. In plants, the ester bond is metabolized, forming the acid, which is usually more phytotoxic. Depending on the herbicide, deesterification also can result in immediate herbicide detoxification, as is the case with thifensulfuron- methyl in certain plant species.
Propanil is the most widely studied pesticide with regard to amide hydrolysis. Rice (Oryza sativa L.) is tolerant to propanil because of high levels of aryl acylamidase, which cleaves the amide bond and is the basis for crop selectivity Propanil resistance is due to enhanced hydrolysis by aryl acylamidase in resistant barnyard grass and resistant jungle-rice (Echinochloacolona) biotypes.
In plants, there is limited literature on the role of phosphatases and sulfatases in pesticide metabolism (Hoagland and Zablotowicz 2001). Nitrile hydrolysis is the main route of metabolism of bromoxynilin wheat and of cyanazine in wheat and potato (SolanumtuberosumL.).
Hydrolysis of the nitrile group produces an amide moiety that is converted to carboxylic acid, which may be subsequently decarboxylated. In plants, the major metabolic route for the phenylcarbamate pesticides CIPC and IPC is aryl hydroxylation and conjugation, rather than hydrolysis of the carbamate moiety.
Aromatic Nitroreductive Processes
Generally, nitroaromatic compounds are transformed to different products in individual plant species. For example, the major metabolite of trifluralin in peanut (ArachishypogaeaL.) is N-depropylatedtrifluralin, whereas in sweet potato (IpomoeabatatasL.), the monoamino- derivativeof trifluralin is predominant.
In plants, glutathione conjugation of pentachloronitrobenzene occurs concomitant with the removal of Cl or NO2. Glutathione-mediated displacement of the nitro group of aromatic compounds has also been described in plants.
It is sometimes difficult to separate biological and chemical xenobiotic reductions because reduction of aromatic nitrogroups, e.g., trifluralin and diphenyl ether herbicides, may be coupled with anaerobic reduction of humic acids or iron reduction.
The conversion of the herbicide acifluorfen to aminoacifluorfen is a common example of an aromatic nitroreduction reaction. There is potential to develop transgenic crops that express a bacterial nitroreductase gene to metabolize diphenyl ether herbicides, thereby providing crop tolerance to these herbicides (Zablotowicz etal.2001).
Pesticide Conjugation Reactions
Carbohydrate and Amino Acid Conjugation
Hall etal. (2001) recently defined pesticide conjugation as the ‘‘metabolic process whereby an exogenous or endogenous natural compound is joined to a pesticide or its metabolite(s) facilitating detoxification, compartmentalization, sequestration, and/or mineralization.’’ Conjugation of pesticides often involves utilization of existing enzymatic machinery and is therefore called a cometabolic process.
Glucose conjugation to pesticides occurs primarily in plants, resulting in several metabolites including O-, S-, and Nglucosides, glucose ester, gentibioside (e.g., 6-O-b-D-glucopyranosyl-D-glucose), and malonyl-glucose conjugates.
The most common glucose conjugates are O-glucosides because pesticide oxidation reactions form hydoxyl groups, which are suitable sites for glucose conjugation. Differential conjugation of 2, 4-D imparts differences of susceptibility in wheat and some broadleaf species.
Many susceptible broadleaf weeds produce glucose ester metabolites, which are readily susceptible to hydrolysis, yieldingphytotoxic 2, 4-D. Conversely, 2,4-D–tolerant wheat rapidly produces amino acid conjugates and O-glucosides, which are stable nonphytotoxic metabolites that are not easily hydrolyzed.
Amino acid conjugation occurs primarily in plants and is very common. Most of the research on amino acid conjugation of pesticides has been conducted on 2, 4-D and twenty amino acids have been found to conjugate with the herbicide.
Uridine diphosphate–glucosyl (UDPG) transferase, anenzyme involved in cellulose biosynthesis, mediates pesticide–glucose conjugation and pesticide–glucose ester conjugation reactions.
As mentioned above, glucose esters of pesticides are cleaved by esterases, often resulting in the release of the pesticide. However, the addition of a second glucose molecule to the glucose ester produces a gentiobiose conjugate, which is not readily hydrolyzed.
Other complex sugar conjugates in addition to gentibioside (two glucose molecules) are glycosides (a glucose and one other sugar, such as arabinose). Pesticide–sugar conjugates can undergo further conjugation with malonate via reaction with malonyl CoA, a common reaction in higher plants.
In tomato (LycopersiconesculentumL.), the herbicide metribuzin is conjugated to glucose, which is subsequently conjugated to malonate, forming the N– malonyl–glucose conjugate.
A range of UDPG transferase activity within various tomato cultivars confers differential tolerance of these cultivars to metribuzin (Smith et al. 1989). Furthermore, increased metribuzinphytotoxicity in all the cultivars was noted under low light conditions.
It was speculated that under low light conditions less glucose and UDPG were produced, thereby reducing conjugation and elevating herbicide phytotoxicity.
Plant Glutathione Conjugation Reactions
Glutathione (g-L-glutamyl-L-cysteinylglycine [GSH]), commonly present in the reduced form, is ubiquitously distributed in most aerobic organisms. Homoglutathione (g-Lglutamyl-L-cystein-b-alanine), a GSH analog, occurs in several legume species.
Although GSH concentrations vary during plant development, GSH is found in relatively high concentrations in most plant tissues. Glutathione is phloem mobile and is degraded by carboxypeptidases and transpeptidases in the cytoplasm and vacuoles. Generally, GSH synthesis is limited by availability of cysteine and hence by the concentration of sulfate ions.
Non-enzymatic GSH conjugation may be important for the metabolism of several herbicides. For example, increased GSH concentrations protects wheat from fenoxaprop injury, and this reaction is considered nonenzymatic because glutathione S-transferase (GST) activity in these plants is low.
However, enzymatic conjugation of xenobiotics with GSH via GSTs is more common than nonenzymatic conjugation. Glutathione-S– transferases are homo- or heterodimer, multifunctional enzymes located in the cytosol, which catalyze the nucleophilic attack of the sulfur atom of GSH by the electrophilic center of the substrate.
More than 50 plant GST gene sequences from 13 plant species have been published. Compared with other plant and bacterial species, corn (Zea mays L.) GST gene enzyme systems have been the most extensively studied.
The role of GSTs and GSH in plants encompasses several major functions. The first is the metabolism of secondary products, including cinnamic acid and anthocyanins. A second function is regulation and transport of both endogenous and exogenous compounds, which are often GS-X tagged for compartmentalization in the vacuole or cell wall (Hatzios 2001).
This is a particularly important aspect for herbicides, anthocyanins, and indole-3-acetic acid. Protection against oxidative stress from herbicides, air pollutants (Sharma and Davis, 1994), pathogen attack, and heavy metal exposure is a third function. Glutathione conjugates and their terminal metabolites are stored in the vacuole or bound to the cell wall.
Formation of Bound Pesticide Residues
Pesticides (mainly conjugated pesticides) are often bound to plant cell walls. Bound pesticide residues are generally considered as those that cannot be extracted with aqueous and organic solvents.
However, a more precise definition has been provided by Skidmore et al. (1998): A bound xenobiotic residue is a residue associated with one or more classes of endogenous macromolecules.
It cannot be dissociated from the natural macromolecule using exhaustive extraction or digestion without significantly changing the nature of the associated endogenous macromolecules.
When studying bound-pesticide residues using radiolabeled pesticides, it is important to differentiate the bound residue containing the labeled xenobiotic or its metabolite from the ‘‘natural label’’ Natural labeling occurs when 14CO2 is released from the mineralized pesticide and is incorporated into the plant cell wall. Natural labeling in plants has been observed with several pesticides.
Furthermore, it is important to know the precise position of the label on the pesticide molecule so that the site of pesticide incorporation into the cell wall can be determined (Sandermann etal. 2001). Digestive treatment with different enzymes such as cellulase, collagenase, pepsin, amylase, and proteases can aid in identifying the nature of pesticide incorporation.
On the basis of reports in the literature, it appears that xenobiotics are incorporated randomly into different cell wall components (Sandermann et al. 2001); however, little is known about the type of linkages involved in this binding.
There is concern about the bioavailability of bound pesticides from plant residues. Phanerochaetechrysosporiummineralized bound chloroaniline and 2,4-dichlorophenol, indicating that these compounds may become bioavailable. The ability of animals to release xenobiotics bound to plant residues is unknown.
Experiments using a ‘‘simulated stomach’’ demonstrated that pesticides were released from plant residues, but only when high concentrations of bound pesticide residues were used. In comparison, only low concentrations of bound pesticide residues are typically present in plant residues (Sandermann et al. 2001).
However, the biological relevance of typically low concentrations of bound pesticide residues is not known. Presently, the U.S. Environmental Protection Agency requires no characterization ofbound pesticide residues if concentrations are less than 0.05 ppm of the parent equivalents or 10% of the total pesticide residue.
If concentrations exceed these levels, determination of the bioavailability based on ‘‘simulated stomach’’ experiments is required. The toxicological nature and bioavailability of bound xenobiotic residues requires continued research to fully assess its impact on human health and theenvironment (Sandermann etal. 2001).
In conclusion, the basis for selectivity of plants to pesticides has been extensively studied during the past 40 years and has provided a wealth of information on diverse biological processes and enzymes in plants.
Understanding the plant enzymatic systems involved in metabolic processes will provide a basis for developing novel, more effective, and environmentally benign herbicides and safeners. One particular strategyis phytoremediation, a process by which plants and their associated microorganisms collectively degrade, detoxify, and remove pollutants.
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