Root Ecophysiology
Detoxification of electrophilic xenobiotics in plants
All organisms are frequently exposed to an array of potentially toxic substances, be they inorganic or organic in chemical nature. These substances may originate naturally from fires, volcano eruptions or processes of biodegradation. They may as well spring from microbial or animal metabolism, and also from the plant’s secondary metabolism (Naumann 1993, Altshuller 1983). They may act as active substances in defense or in allelopathic reactions. Furthermore, increasing industrialization has provided two novel sources of foreign compounds: first by the invention and use of agrochemicals for the protection of crops and the control of pests and weeds, and second by the emission of organic xenobiotics in the process of chemical production of goods or the use of synthetic chemicals. The latter compounds of solely anthropogenic origin represent a threat to our environment as they are emitted without any control. For plants, the situation is especially awkward as they root in the ground and are bound to survive on a certain site. Hence they have to rely on effective detoxification mechanisms in a special manner.
The uptake of xenobiotics from whatever polluted medium, i.e. air, water or soil, follows the laws of phase distribution and diffusion and hence plants have only limited possibilities to avoid accumulation of foreign compounds in their tissue and the connected detrimental consequences. In the context of glutathione mediated detoxification processes, especially those foreign compounds bearing electrophilic sites are of major importance.
Various electrophilic xenobiotics, i.e. compounds with centers of low electron density that can accept an electron pair to form a covalent bond, exhibit the tendency to react spontaneously with nucleophilic sites (i.e. centers of electron richness, nonbonded pairs of electrons or p bonds) of biomolecules. Thus, electrophilic xenobiotics may be highly dangerous to the cell, because they are able to bind to proteins and genetic material, i.e. DNA and RNA and thereby disturb metabolic networks.
Reactivities between nucleophilic biomolecules and electrophilic xenobiotics (from Coleman et al. 1997, with modifications).
Nucleophilic site | Softness/Hardness | Electrophilic site |
|---|---|---|
sulfur in cysteinyl residues of proteins or GSH | Soft | polarized double bonds, aldehydes |
sulfur in methionyl residues of proteins |
| epoxides, alkyl sulfates, alkyl halides, stained ring lactones |
amino groups in proteins (Arg, Lys, His) |
|
|
amino groups of purines in DNA and RNA |
| arylcarbonium ions |
oxygens of purines and pyrimidines |
| benzylic carbonium and nitrenium ions |
phosphate oxygen of RNA and DNA | Hard | alkylcarbonium ions |
In this view, an organism would need some prerequisites to perform detoxification reactions relying on glutathione:
Prerequisites for glutathione dependent detoxification:
- electrophilic centers on the xenobiotic to be attacked
- adequate supply of glutathione or its analogues
- glutathione S-transferase with respective substrate specificity
Unusual features of glutathione
Bearing two carboxylic acid groups, one amino group and one thiol group and having two peptide bonds, glutathione is highly hydrophilic. Glutathione conjugation of hydrophobic electrophilic compounds thus leads to a loss of their lipophilicity by converting the parent compound into an amphiphilic product with a bulk hydrophilic region and the nonpolar hydrophobic region. This change of physico-chemical properties impedes the mobility of the foreign compound and inhibits further partitioning into membranes as well as the diffusion between compartments. Even more important, the products of this reaction are subject to ionisation at cellular pH and definitely restricted in their availability to the cells and tissues.
The three-phases concept of detoxification
Some xenobiotic compounds have to be activated for conjugation with glutathione or GST mediated conjugation. Most if not all conjugation products then undergo metabolism, transport and long term storage or excretion from the living cells in plants. The parallels with animal metabolism of xenobiotics in intestine, liver and kidney have been pointed out early by Richard Shimabukuro (1971) from the USDA Bioscience Research Laboratories in Fargo, ND. His ideas have been confirmed later by several authors (Lamoureux and Rusness 1989, Lamoureux et al. 1991, Coupland 1991, Coleman et al. 1997, Schröder 1997) and have led to the „green liver concept“ favored by Sandermann and coworkers (Sandermann 1994, Sandermann et al. 1997). All concepts have in common the option of xenobiotic activation by P450 monooxygenases and related enzymes in a first step designated phase I, the detoxification in the true meaning of the word in phase II and the metabolism, breakdown or final storage in phase III (Figure 1).
Figure 1. The three phase model of detoxification in animals and plants (adapted from Shimabukuro 1976, Coupland 1991, Sandermann et al. 1997). In phase I xenobiotic molecules are activated via oxidation, reduction or hydrolysis, and in phase II detoxification is achieved by conjugating biomolecules, e.g. sugars, amino acids or glutathione to the activated sites. Compounds with sufficiently high electrophilicity may be conjugated without activation. Phase III is characterized as cleavage, secondary conjugation and metabolization of conjugates and may include compartimentation into the vacuole, the apoplast or the cell wall.
In addition to internal storage processes, excretion into the soil with root exudates or into the atmosphere after volatilization (Lamoureux et al. 1993) may be a significant phase III step in plants for some compounds as has recently been shown for metabolites of the diphenylether herbicide fluorodifen.
Of all phases, phase II has the largest effect on the effective toxicity of foreign compounds taken up by the cell. It has to be mentioned that for a large number of xenobiotics, especially after hydroxylation reactions in phase I, sugar conjugation by glycosyltransferase action is the detoxification step. This reaction is very effective, however it has frequently been observed to be reversible. Numerous glycosidases exist that may become active under certain conditions in the cell and liberate the hydroxylated parent xenobiotic by cleaving the glycosyl residue. A way to overcome this problem of retarded toxicity seems to be the formation of bound residues outside of the cytosol, in the cell wall.
For glutathione conjugation, the situation is different. The mechanism of glutathione binding to a xenobiotic includes the cleavage of reactive centers, mostly halogens, from the xenobiotic in exchange with the cysteinyl sulfur. Even if cleavage of this bond occurred (see below) the resulting product would be devoid of the electrophilic center of the parent and would therefore not be as reactive as before. Furthermore, experimental evidence has shown that the sulfur bond to the molecule is very stable because cleavage of conjugates includes destruction of the cysteinyl moiety. Thus the former xenobiotic, even if liberated (by metabolic activity), will not have the chemical nor the toxicological characteristics of the original compound, i.e. the electrophilicity will be lowered. These considerations are valid for most of the glutathione dependent conjugation reactions reported, except for a special type of reactions on compounds with reactive carbon-carbon double bonds neighbored by an electron withdrawing group (Talalay et al. 1988). The conjugation on these bonds is a so called Michael reaction and leads to a labile conjugate that may be sensitive to pH changes (Ishikawa 1987).
