Phases of herbicide metabolism

Herbicide metabolism has been well documented in the last two decades and much progress has been made in our understanding of the enzyme systems involved and where they act in the plant cell. The four phases commonly observed are as follows:

Bioactivation

Phase I Metabolic attack

Phase II Conjugation

Phase III Sequestration

Some herbicides have been shown to undergo bioactivation within plant cells, where a pro-herbicide is converted to a phytotoxic agent by the action of plant enzymes. Before this activation they may be less or non-phytotoxic, so the plant can be instrumental in manufacturing the substance that will eventually kill it. Bioactivation can involve removal of chemical groups that have aided in herbicide uptake and this can have the added benefit of trapping the herbicide within the cell. Herbicide detoxification is achieved by key enzymes that carry out two major functions. First, they alter the chemical structure of the herbicide to render it biologically inert. Second, these reactions serve to increase both the reactibility and the polarity of the herbicide, so that is can be removed from the cytoplasm and either stored in the vacuole or bound to the cell wall. Metabolically, this is achieved by introducing or uncovering polar groups (Phase I metabolism). In some cases polar groups may already be in place in the original herbicide structure, in which case Phase II metabolism can be carried out without the need for Phase I reactions. In other cases, conjugate formation may be reversible and this may serve to store phytotoxic molecules.

4.2.1.1 Bioactivation

Bioactivation is the process whereby 'pro-herbicides', often herbicidally inactive molecules, are enzymatically converted to phytotoxic compounds in the plant cell. Many herbicides are therefore formulated as inactive hydrophobic esters to enable them to penetrate the waxy leaf cuticle. Ester hydrolysis then reveals the biologically active acid or alcohol groups. Examples include the conversion of bromoxynil octanoate to bromoxynil, and diclofop-methyl to diclofop acid (Figure 4.1). Both the aryloxyphenoxypropionate graminicides, such as fenoxaprop-ethyl, and the phenoxy-carboxylic acids, such as 2,4-DB, are rapidly de-esterified in both crops and weeds. De-esterification may, in some cases, inactivate herbicides, as in the metabolism of sulphonylurea esters, such as chlo-rimuron-ethyl, in which the de-esterified product is herbicidally inactive. Another example is the conversion of EPTC to the herbicidally active sulphoxide derivative. Imazamethabenz-methyl may also be regarded as a pro-herbicide. In susceptible weeds, hydrolysis results in a potent inhibition of branched chain amino-acid biosynthesis, whereas hydroxylation of the intact ester occurs in resistant maize and wheat. Bioactivation of DPX-L8747 by N-dealkylation in susceptible species leads to an active herbicide, whereas in resistant crops hydroxylation following the formation of a glutathione conjugation of the intact pro-herbicide leads to non-toxic metabolites. These examples demonstrate how bioactiva-tion may be a mechanism of selectivity between crop and weed. Opening of the oxadio-zolidine ring of methazole to form 1-(3,4-dichlorophenyl) urea and N-demethylation of bromoxynil octanoate (inactive)

bromoxynil (active)

Br bromoxynil octanoate (inactive)

Br bromoxynil (active)

diclofop-acid (active)

Figure 4.1 Bioactivation of inactive pro-herbicides to active molecules.

diclofop-acid (active)

Figure 4.1 Bioactivation of inactive pro-herbicides to active molecules.

pyridazinone to form a potent phytoene desaturase inhibitor, further demonstrates the wide range of chemical reactions that can lead to bioactivation. In the case of the bioherbicide bialaphos, a tripeptide obtained from Streptomyces, metabolic cleavage results in the release of glufosinate, which is an important herbicide in its own right. Interestingly, resistance to the herbicide triallate in a biotype of Avena fatua has been demonstrated to be due to a reduced ability to convert triallate to the phytotoxic product triallate sulphox-ide. This is the only reported instance of resistance being due to an inability of a weed to bioactivate a herbicide.

Cummins et al. (2001) found a large number of diverse proteins in wheat capable of hydrolysing herbicide esters and these activities differed from those in competing grass weeds. Indeed, crude extracts from black-grass plants were more active in ester hydrolysis than wheat. They purified a 45 kDa esterase from wheat that could bioactivate bromoxynil octanoate, but showed no activity towards diclofop-methyl. Conversely, esterase activity towards diclofop-methyl was higher in the weed than the crop. Their observations support the rapid bioactivation by ester hydrolysis of graminicides in grass weeds, contributing to their selectivity. The location of this esterase activity is thought to be the cell wall.

4.2.1.2 Metabolic attack

This phase of metabolism aims to introduce or reveal chemically active groups, such as -OH or -COOH, which can undergo further reactions. The most common way in which plants attack herbicides is by hydroxylation of aromatic rings or of alkyl groups by a family of enzymes known as the cytochrome P450 mono- or mixed function oxidases (P450s).

The P450s are a very large family of enzymes now thought to be the largest family of enzymatic proteins in higher plants. They all have a haem porphyrin ring containing iron at a catalytic centre. These enzymes are responsible for the oxygenation of hydrophobic molecules, including herbicides, to produce a more reactive and hydrophilic product. The reaction utilises electrons from NADPH to activate oxygen by an associated enzyme, cytochrome P450 reductase. One atom from molecular oxygen is incorporated into the substrate (R), while the other is reduced to form water:

The enzymes are located on the cytoplasmic side of the endoplasmic reticulum and are anchored by their N-terminus (Figure 4.2). They are found in all plant cells but in very low abundance. This, coupled with their lability in vitro, has meant that they are difficult to study biochemically. All P450s have a highly conserved region of 10 amino acids surrounding the haem group and it is this region that is responsible for the binding of O2, its activation and the transfer of protons to form water. The rest of the P450 amino acid sequences are highly variable and this probably explains the wide variety of reactions and substrate specificity shown by this enzyme superfamily.

Their name is derived from the maximum absorbance at 450 nm when the reduced enzyme is bound to the inhibitor carbon monoxide, and the 'P ' signifies protein. This inhibition by carbon monoxide is typically overcome by light. These features are all used in setting criteria for the involvement of P450 enzymes in herbicide metabolism. These criteria are:

• requirement for NADPH,

• association of enzyme activity with the microsomal fraction produced by centrifugation at 100,000 x g, enriched in the endoplasmic reticulum,

• inhibition by CO, which is reversible by light,

• inhibition by anti-reductase antibodies, and

• inhibition of in vitro activity by known P450 inhibitors, including aminobenzotriazole, paclobutrazole, piperonyl butoxide and tetcyclasis.

substrate

haem

Figure 4.2 A diagrammatic representation of plant P450 and P450 reductase enzymes on the endoplasmic reticulum (after Werck- Reichhart et al., 2000).

substrate haem

Figure 4.2 A diagrammatic representation of plant P450 and P450 reductase enzymes on the endoplasmic reticulum (after Werck- Reichhart et al., 2000).

The P450 proteins are between 45 and 62 kDa in size. While their amino acid sequences may vary considerably, their three-dimensional structure is highly conserved, especially in the haem-binding region. The haem binds to the protein at a cysteine residue and the flanking sequence (Figure 4.3) is a characteristic of all P450s.

The conserved oxygen-binding sequence is about 150 residues upstream from the haem and consists of Ala or Gly-Gly-X-Asp or Glu-Thr-Thr or Ser. In both haem- and oxygen-binding sequences, X denotes any other amino acid.

When these conserved sequences were used to study P450s in plants, a surprisingly large number and diversity of P450s was found. Indeed, more than 500 plant P450 genes are now known in over 50 families, indicating that the P450s are the largest group of plant proteins. The precise roles of the proteins encoded by these genes is, however, largely unknown.

A nomenclature has been designed for P450 genes based on the identity of the amino acid sequences of the proteins they encode (Figure 4.4). The genes have been numbered in chronological order depending on their date of submission to the P450 nomenclature committee (http://drnelson.utmem.edu/CytochromeP450.html).

Typical families are numbered from CYP71 to CYP99. For example, CYP71 C6v1 in wheat is inhibited by glyphosate and CYP76B1 catalyses the dealkylation of the phenylurea herbicides (Wen-Sheng et al., 2005).

The discovery of new P450 genes continues in plants. In contrast, only about 50 P450 genes in 17 families have been described in humans. So why are there so many P450s in plants? The answer seems to be that they play a very wide role in plant secondary metabolism. They have been shown to be involved in the biosynthesis and metabolism of a wide variety of compounds, including terpenes, flavonoids, sterols, hormones, lignins, suberin, alkaloids and phytoalexins. They are also induced by pathogen attack, xenobiotics and by light-induced stress, unfavourable osomotic conditions, wounding and infection.

It is currently believed that herbicide molecules also fit the active sites of these P450s involved in biosynthesis, suggesting a broad diversity of substrate selectivity.

Regarding their roles in herbicide metabolism, much remains to be done to establish substrate specificity and both the molecular and the metabolic regulation of these

Gly - Cys - X - Arg - X - Gly - X - X - Phe Figure 4.3 The haem-binding sequence that is characteristic of all P450s.

CYP XX YY ZZ

lilt denotes family of genes with subfamily sharing number indicates

P450 gene at least 40% at least 55% isoforms sharing similarity similarity 95% or greater similarity

Figure 4.4 Nomenclature for naming P450 genes.

important enzymes. Such understanding will be invaluable in predicting and elucidating herbicide selectivity, as well as in the discovery and design of new selective herbicides.

The main reactions catalysed by P450s are shown in Figure 4.5. In herbicide metabolism these are hydroxylation and dealkylation, which progresses via a hydroxylation step. Examples of herbicides metabolised by P450s in plant systems include sulfonylureas (including primisulfuron, nicosulfuron, prosulfuron, triasulfuron and chlorimuron), substituted ureas (chlorotoluron, linuron), chloroacetanilides (metolachlor, acetochlor), tria-zolopyrimidines (flumetsulam), aryloxyphenoxypropionates (diclofop), benzothiadiazoles (bentazon) and imidazolinones (imazethapyr). Selectivity to herbicides can be due to ability of the crop to metabolise herbicides via P450s, an ability that may not be possessed by susceptible weeds. In some cases, however, this metabolism is not enough to prevent crop damage, either because of low rates of P450 metabolism or phytotoxicity of products produced by these reactions. Crop damage may only be prevented if reactions from Phase II (conjugation) are successful in carrying out further detoxification.

Some Phase I reactions may be catalysed by peroxidases (E.C. 1.11.1.7) which are commonly found in leaves at high concentrations, being able to catalyse oxidations using hydrogen peroxide. They are currently thought to be involved in proline hydroxy-lation, indole acetic acid (IAA) oxidation and lignification, and have been implicated in the metabolism of aniline compounds produced in the degradation of phenylcarbamate, phenylurea and acylaniline herbicides.

4.2.1.3 Conjugation

In this phase of herbicide metabolism, the molecule becomes conjugated to natural cell metabolites, such as amino acids, sugars, organic acids or the tripeptide glutathione (y-glutamyl-cysteinyl-glycine). This has the effect of both further reducing phytotoxicity and increasing the solubility of the herbicide or its metabolite, which may also serve to target the conjugate to the vacuole.

The most widely studied conjugation reaction in relation to herbicide detoxification is that of glutathione conjugation, carried out by the enzyme family glutathione S- transferases (GSTs), E.C. 2.5.1.18. Glutathione is abundant in plants, often exceeding 1 mM concentration in the leaf cell cytoplasm, where it functions as a scavenger of free radicals, protecting photosynthetic cells from oxidative damage. As GSTs are a large group of similar enzymes found in all eukaryotes, differences in the spectrum of GSTs present plays an important role in selectivity of herbicides. GSTs have a range of endogenous functions involving their abilities to detoxify and act as redox buffers.

The GSTs are abundant, soluble enzymes of about 50 kDa, each composed of two subu-nits of equal size, containing an active site located in the N-terminus that binds glutathione and is highly conserved in all GSTs. Herbicides and other xenobiotics are bound at the hydrophobic C-terminal half of the subunit. This site varies considerably and accounts for the differing specificity of GSTs towards herbicides.

Arabidopsis thaliana has at least 30 distinct GST genes and their nomenclature is complex. The subunits have been classified on the basis of their amino acid sequence and similarities in gene structure. The classes are termed phi (F) zeta (Z), theta (T) and tau (U) and are used in conjugation with the species name, for example:

epoxide

Figure 4.5 The main reactions carried out by cytochrome P450 monooxygenases (P450s).

In Zea mays:

Zm GST F1

F2 F3 U1 U2 U3

In Arabidopsis thaliana:

At GST T1

F1 U1

In Alopecurus myosuroides:

Am GSTU1 F1

Why are there so many GSTs in plants and what are their physiological roles? Although often cited in the literature, the involvement of GSTs in the conjugation of secondary metabolites remains far from clear. A function in shuttling anthocyanin pigments to the tonoplast for vacuole uptake appears likely.

Plants exposed to environmental stress or infection show elevated expression and activity of GSTs, suggesting a role in maintaining cellular homeostasis following oxidative stress. Indeed, herbicide resistant black-grass has been shown to have higher activity of GST (Reade et al., 2004).

Edwards and Dixon (2000) consider that GSTs are well placed to use physiologically high cytoplasmic concentrations of glutathione (0.2-1.0mM) to conjugate electrophilic herbicide residues effectively. As the conjugates can inhibit GST activity, they are actively transported out of the cytoplasm into the vacuole by the ATP-binding cassette (ABC)-transporters. When the ABC-transporter gene from Arabidopsis was expressed in yeast, Lu and colleagues (1997) demonstrated the uptake of S-metolachlor-glutathione, confirming the involvement of these transporters across the tonoplast membrane.

Once in the vacuole, a specific vacuolar carboxypeptidase hydrolyses the glycine residue and the dipeptide conjugate is re-exported to the cytoplasm. The glutamate group is removed by a glutamyl transpeptidase and the remaining cysteinyl derivatives are further transformed by N-malonylation and further oxidation to a complex range of more polar products.

GST activity was first demonstrated in plant tissue against atrazine in maize extracts in 1970. Since this observation, GST activity against a wide variety of herbicides has been reported (Table 4.1). As with other conjugate types, glutathione conjugation can be carried out against the parent herbicide if an appropriate conjugating group is present, or can follow Phase I metabolism. An example of the latter is the conjugation of glutathione with thiocarbamates, only after they have undergone conversion to their corresponding sul-phoxides. Crops are often reported to possess higher GST activities against herbicides than susceptible weeds and this might offer some degree of selectivity between crop and weed. Activity against chloroacetamides (maize, wheat, sorghum, rice), oxyacetamides (maize), atrazine (maize, sorghum), fenoxaprop, fluorodifen, flupyrsulfuron-methyl, dimethenamid (all wheat) and the sulphoxide metabolite of EPTC (sorghum) have all been reported. In soybean, homoglutathione is found in place of glutathione. Conjugations utilising this against several chloroacetanilides, the diphenyl ethers acifluorfen and fome-safen, and the sulphonylurea chlorimuron-ethyl are all reported in this crop. In addition to selectivity, GSTs have also been implicated in playing a role in herbicide resistance in

Table 4.1 Examples of some herbicides metabolised by glutathione S-transferases in various plant systems.

Chemical family

Examples

Chloroacetamides

Alachlor, acetochlor, metolachlor, pretilachlor

Triazines

Atrazine

Aryloxyphenoxypropionates

Fenoxaprop

Thiocarbamates

EPTC

Diphenyl ethers

Acifluorfen, fomesafen

Sulfonylureas

Clorimuron-ethyl, triflusulfuron-methyl

a variety of weeds. In black-grass, biotypes resistant to chlorotoluron and fenoxaprop-ethyl demonstrated approximately double the GST activity of susceptible biotypes. This suggests that GSTs, as well as P450s, may play a role in enhanced metabolism resistance in this species. In velvetleaf (Abutilon theophrasti), resistance to atrazine has also been demonstrated to be due to higher conjugation of this herbicide to glutathione.

Another commonly encountered Phase II reaction in plants is conjugation with glucose, catalysed by the glucosyltransferases (EC 2.4.1.71) utilising uridine diphosphate glucose (UDPG) as the glucose donor. Once conjugated, the glucose may undergo a further Phase II reaction by 6-O-conjugation with malonic acid, catalysed by the malonyl-CoA-dependent malonyltransferases. These Phase-II metabolites then undergo ATP-dependent transport into the vacuole.

Interestingly, Pflugmacher and Sandermann (1998) found O-,N- andS-glucosyltransferase activity to be very widely distributed throughout the plant kingdom - not solely confined to higher plants, but even in marine macroalgae. Indeed, they hypothesised that this activity in - -ower - plants may make an important contribution to the detoxification of xenobiotics in the global environment.

Finally, acidic herbicide molecules such as the synthetic auxin phenoxyacetic acids can be conjugated to the amino acids glutamine, valine, leucine, phenylalanine or tryptophan, although the enzymology of these reactions remains obscure. As an example, crop plants are able to rapidly detoxify the photosynthetic inhibitor bentazone by rapid aryl hydroxy-lation followed by conjugation to glucose. Susceptible weeds appear unable to metabolise the parent herbicide and phytotoxicity is observed.

A 200-fold margin of selectivity between rice and Cyperus serotinus has been attributed to this metabolic route. Similarly in soybean, where an 8-hydroxy derivative has been detected, Leah et al. (1992) isolated and purified two glucosyltrans-ferases from tolerant soybean that were capable of glycosylating 6- hydroxybentazone (Figure 4.6). This soluble enzyme had a relative molecular mass of 44.6 kDa with binding constants for kaempferol and 6-hydroxybentazone of 0.09 and 2.45 mM, respectively. They also found a membrane-bound enzyme, whose primary substrate was p-hydroxyphenylpyruvic acid, with a relative molecular mass of 53 kDa and binding constants of 0.11 and 1.96 mM for p-hydroxyphenylpyruvic acid and 6-hydroxy-bentazone, respectively. These findings, and those subsequently shown by others, imply an overlapping specificity of aryl hydroxylated herbicides and the synthesis and storage of secondary metabolites.

H 6-hydroxybentazone

glucosylhydroxybentazone Figure 4.6 Metabolism of bentazone in tolerant plant species.

glucosylhydroxybentazone Figure 4.6 Metabolism of bentazone in tolerant plant species.

4.2.1.4 Sequestration

Compartmentalisation of a herbicide metabolite appears to take place in much the same way as products of plant secondary metabolism are moved for storage. The place for storage is either the vacuole or in association with the cell wall. Identification of a membrane-bound glutathione-dependent ABC pump in the vacuolar membrane suggests that Phase II conjugation to glutathione or malonate might serve to facilitate the movement of metabolites and could be considered as a way of 'tagging' molecules for movement into the vacuole.

The processing and vacuolar import of herbicide conjugates in plants is a two- step process, first involving glucosylation and then derivatisation of the sugar with malonic acid. The significance of this reaction is not entirely understood, though malonylation appears to act as a tag, directing the conjugates for vacuolar import.

The malonylation reaction is carried out by malonyltransferases which can conjugate compounds containing amino (^-malonylation) or hydroxyl (O-malonylation) residues.

Malonylation also appears to prevent digestion by glucosidases and may also facilitate conjugate transport across the plasma membrane. For example, glucosidic conjugates of pentachlorophenol formed in soybean and wheat undergo malonylation, and 3,4-dichloroaniline undergoes ^-malonylation in soybean and wheat. In each case, the transport of the newly formed conjugates was shown to be routed towards the vacuole. Although malonylation of glucosides is important in directing their importation into the vacuole, it is clear that the glucosides themselves can undergo vacuolar deposition.

Once conjugates are situated in the vacuole, sequential removal of peptides from glu-tathione is carried out by peptidases. This results in the metabolite being conjugated to glutamylcysteine and possibly just to cysteine. It is postulated that this allows for recycling of amino acids back to the cytoplasm and in addition may prevent the conjugated metabolite from being exported back there, as it no longer is a full glutathione conjugate. This pumping mechanism may have the additional benefit of stopping the build-up of glutath-ione conjugates from inhibiting cytoplasmic GST activity, as some conjugates have been demonstrated to be powerful competitive inhibitors of GSTs. Once the metabolite conjugate has entered the vacuole it may be further metabolised, stored there or excreted across the plasma membrane to the extracellular matrix. Transport of glucosylated herbicides into the vacuole has also been reported. This is ATP-requiring, so is also active transport. It appears that the membrane pump carrying this out is distinct from the glutathione system (Joshua et al., 2007).

Most herbicide metabolites eventually become associated with the insoluble compartments of the cell, bound to lignin or polysaccharides. In these forms they can be released by enzymic hydrolysis, and both hydroxylated and unaltered forms have been detected. It is thought that this process can occur by covalent linkage.

While conjugates usually represent the end-product of herbicide metabolism, they should not be regarded as totally inert. Some evidence exists for the hydrolysis of the glucoside to regenerate an active molecule, and so the conjugate may be regarded as a reservoir of potential activity. Such regeneration depends on the nature of the glycoside linkages involved and their proximity and susceptibility to the action of P-glucosidase.

An overview of the different phases of herbicide metabolism in a plant cell is presented in Figure 4.7 .

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  • PETTERI P
    What is a conjugated herbicide?
    3 years ago
  • sanna-leen
    How herbicide metabolism in plants occurs?
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