Mode of action studies

The term 'mode of action' may be defined as the complete sequence of events leading to plant injury, and therefore includes all areas of interaction between a herbicide and a crop or test species. A great many features contribute to successful weed control and may be categorised into areas of herbicide uptake, movement and metabolism (Figure 2.4).

The term 'primary or target site' is commonly used to describe the biochemical location at which a herbicide may potently inhibit an important process. This site is expected to show the fastest response to the herbicide and should also be the most sensitive site to yield a commercially useful effect. This precise site of action is therefore the location of a molecular interaction which triggers a series of secondary events leading to the death of a target weed. An example of such a target site is the D1 protein in the thylakoid membranes of the chloroplast. Many herbicides bind to this protein and so inhibit photosynthetic electron flow through the thylakoid. Consequently, active oxygen species are generated which cause plant death by photo-oxidative damage (Chapter 5- . The D1 protein is a well - known example of a target site with a specific and strategic membrane location. Other examples of known target sites include natural receptors to plant growth substances (e.g. the auxin--ype herbicides, Chapter 7- , and specific enzymes in the biosyntheses of lipids and amino acids (Chapters 8 and 9, respectively).

There is no proven or simple route to discover the target site of a new herbicide. On the contrary, the investigator requires an appreciation of the vagaries of plant metabolism, an enjoyment of detective work and a large measure of good fortune! A sequential investigation is often followed to establish a target site, as described below. However, this may give the false impression of standardised procedures. Although such an approach is generally sound and valid, it should be noted that the investigator may uncover uncharted areas of plant metabolism, in which the novel herbicide may be regarded as







Plant death

Action at target sites


Changes in cell structure and function

Molecular fate

Figure 2.4 Herbicide mode of action.

a new and powerful probe. In this way photosynthetic inhibitors have allowed the molecular dissection and characterisation of Photosystem II (Chapter 5), and, as a further example, our understanding of aromatic amino acid biosynthesis was greatly aided from the study of glyphosate action (Chapter 9).

A sequential investigation to discover the target site of a herbicide is as follows:

1 Symptom development in target species.

2 Structural analysis of sensitive tissues.

3 Monitoring of key physiological processes.

4 Biochemical studies.

Detailed observations of symptom development provide the starting point for her-bicidal detective work. If these observations are recorded daily the speed of response is quickly established and the most sensitive tissues identified. In addition, the development of secondary effects may have useful diagnostic value, as will the addition of ' standard' herbicides to the study. Thus, if symptoms are similar to those produced by existing herbicides, a shared target site may be implied. For example, photosyn-thetic inhibitors typically induce leaf chlorosis before necrosis, and auxin- type herbicides cause characteristic twisting and bending effects in young, elongating tissues of susceptible plants. In the case of soil-applied herbicides, the emergence of ' white' cotyledons is a good indication of the inhibition of carotenoid biosynthesis by the so-called 'bleaching' herbicides. These observations may be followed by ultrastructural comparisons of sensitive tissues from treated and untreated plants. The progressive development of abnormalities in both extracellular and intracellular organisation is a powerful indicator of which metabolic compartments are involved in herbicide action. Isotopic tracers and autoradiography may be used at this stage to pinpoint this cellular site with precision.

The clues generated from the visual and microscopic assessment of treated tissues may then be used to monitor key physiological processes in the presence or absence of the herbicide. Such studies point to the most likely areas of inhibition in the discovery of the target site. For example, photosynthesis and respiration may be measured in intact tissues, or isolated chloroplasts or mitochondria using oxygen electrode techniques. In recent years, many groups and companies have developed model systems for such studies, including the use of isolated cells and protoplasts from target species, cell cultures and green algae. In general, various macromolecular syntheses can be examined in these systems, especially by monitoring the incorporation of radiolabelled precursors into stable products, including leucine into protein, thymidine into DNA, uracil into RNA and acetate or mevalonate into lipids. In addition, physiological viability may be assessed using fluorescent stains, and membrane integrity and function examined by measuring potassium flux. In each case, comparison with herbicide standards should be encouraged and both time-courses and dose-responses should be established to determine the precise sensitivity of the process to herbicidal inhibition.

Once a likely target pathway is indicated, a sequence of reactions is examined in more detail. Initially, concentrations of pathway intermediates should be investigated, both in the presence and in the absence of the herbicide. If one compound accumulates in the presence of the inhibitor, then this is good evidence that the target site of an enzyme has been found. For example, glyphosate treatment results in accumulation of shikimate 3-phosphate, implying the inhibition of 5-enoyl-pyruvyl shikimic acid 3-phosphate (EPSP) synthase, and the inhibition of protoporphyrin oxidase by acifluorfen is pinpointed by the accumulation of protoprophyrin IX in treated plants. Such an accumulation of a metabolic intermediate implies that further key molecules are not being formed. This can be confirmed by studying whether the addition of the key metabolite is able to overcome the inhibition. In this way the inhibition of branched-chain amino acid biosynthesis by the sulphonylureas and imidazalinones can be overcome by the addition of leucine, isoleucine and valine. Additional proof that a true target site has been discovered may come from further studies with mutants. Probably the best-documented example of such mutagenesis is the herbicide site of the D1 protein in the chloroplast thylakoid. That a single substitution at position 264 from serine to glycine is sufficient to cause total resistance to atrazine in some weeds is surely powerful evidence of a target site! Further studies may then focus on the chemical and biochemical properties of the target site. For instance, the kinetics of the inhibition and the precise mechanism of inhibition may be investigated. Such studies are not only of academic value, but are also central to our understanding of herbicide selectivity at the molecular level and may establish precise structure-activity relationships for the development of molecules with optimal activity. Additionally, site-directed mutagenesis may then be employed to create crop tolerance.

In a recent review, Grossmann (2005) has described a functional array of bioassays conducted at BASF, Limburgerhof, aimed at diagnosing the mode of action of a new herbicide molecule. These assays are designed to differentiate between the distinct responses of complex structures (plant, tissue, meristematic cell, organelle), developmental stages, types of metabolism and physiological processes. He has coined the term 'physionomics' to describe this physiological profiling as providing the first clues to mode of action. This term follows the use of other '-omics' technologies in studying herbicide discovery and mode of action:

Functional Genomics: Generating mutants and screening them for functional gene identification.

Transcriptomics: Profiling gene expression utilising DNA microarrays and RNA extraction.

Proteomics: Protein profiling by gel electrophoresis of extracted proteins. Metabolomics: Metabolic profiling using metabolite extraction and separation by gas chromatography / liquid chromatography-mass spectrometry. Physionomics: Physiological profiling following functional bioassays.

Biochemical studies are often routinely performed in cell-free systems on enzymes of fungal or bacterial origin. Several dangers are implicit in this in vitro route, and particular care is needed not to extrapolate results obtained in vitro to predict in vivo activity in the whole organism. Thus, the perfect herbicide in vitro may have negligible practical value if it cannot reach its active site. Indeed, many a promising, lipophilic candidate has not been developed further owing to its being confined to the cuticle and not entering the weed! To this end, increasing use is being made of the physicochemical properties of a molecule that will allow a prediction of its systemicity and stability in both target species and the environment. These include the octan-1-ol / water partition coefficient (Kow), and the dissociation constant (pKa). No single measure of organic phase/water distribution can

Table 2.9 Herbicide systemicity and log Kow

log Kow



3 to >6 (lipophilic)



Xylem mobile

triazines, phenylureas

diflufenican, diphenylethers

Both xylem and phloem mobile

glyphosate, aminotriazole, glufosinate

auxin-type herbicides, sulphonylureas, imidazolinones, sethoxydim

be predicted for herbicides because of the wide variety of organic phases within a plant, such as hydrocarbon waxes, triglycerides, proteins, lignins or even carbohydrates. However, Briggs and colleagues (Bromilow et al., 1986I have found that the partition coefficient can give a good prediction of systemicity. For acids and bases the dissociation constant of the chemical and the natural pH of the various plant compartments will also determine the proportion of ionised and non-ionised forms present. Ionisation decreases log Kow and so can have a dramatic effect on the movement of a compound in a plant. Most phloem-mobile, systemic compounds are therefore weak acids with log Kow in the range of -1 to 3, but the immobile soil-applied herbicide diflufenican has a log Kow value of 4.9 (Table 2.9).

Metabolism of the herbicide within a plant may also have a profound effect on movement. Metabolism in general attempts to reduce lipid solubility and prevent toxic action. For example, aryl hydroxylation may be expected to cause increased mobility from the parent molecule (pKa change from neutral to 10), and its subsequent conjugation to glucose will create a less mobile glucoside (pKa change from 10 to neutral) that becomes subject to further compartmentation or inactivation within the cell (Bromilow et al., 1986). Figure 2.5 presents an overview of how mobility may be related to log Kow and pKa.

Mode of action studies greatly aid, and in many cases totally explain, the selectivity shown by many herbicides. Indeed, susceptibility, tolerance, and resistance are being increasingly defined at the metabolic level. Selectivity may of course be due to differential leaf interception, retention, or uptake, but once inside the plant several metabolic criteria are now evident. Generally, selectivity may be achieved at several steps as suggested in Figure 2.6 .

Many herbicides are themselves inactive (Figure 2.6A) and need to be metabolically activated before phytotoxicity is observed. Thus, paraquat and diquat are activated by light in the thylakoid to generate toxic active oxygen species (Chapter 5); the butyl esters of MCPA and 2,4iD are converted to active acids in susceptible species by P-oxidation (Chapter 7), and some graminicides, such as the aryloxyphenoxypropionates, require conversion from ester to acid for optimal activity (Chapter 8). Active herbicides may be metabolised before they can reach their target site (Figure 2.6B).

Many enzymes, often mixed-function oxygenases, have been implicated in herbicide metabolism studies. Maize and sorghum contain a high concentration of glutathione S-transferase, so that atrazine is conjugated and detoxified before it reaches its thylakoid

Figure 2.5 Relationship between herbicide mobility, log Kow and pKa ( after Bromilow et al., 1986 with permission).

Inactive herbicide


C Target site

Figure 2.6 Herbicide metabolism in relation to selectivity. Points A-D are stages where selectivity may be achieved.

Immobilisation of herbicide in vacuole or at cell wall


Active herbicide

C Target site

Plant death

Figure 2.6 Herbicide metabolism in relation to selectivity. Points A-D are stages where selectivity may be achieved.

site of action, wheat readily hydroxylates chlorsulfuron and the metabolites are inactivated by conjugation with glucose. Similarly, soybean selectivity hydroxylates and glycosylates bentazone; metamitron is selectively deaminated in sugar beet, and diuron is initially demethylated in some species prior to inactivation, again by hydroxylation and glycosylation.

Herbicide metabolites may not always be inactive. On the contrary, evidence is accumulating that glycosyl ester formation may be reversible in many instances. Thus, conjugates stored in the vacuole or bound to cell wall components may be regarded as herbicide reservoirs, which may be called into play at a later stage.

Selectivity may also be achieved at the target site either by differential binding or inactivity (Fig. 2.6C). For example, the spectacular selectivity shown by the aryloxyphe-noxypropionate and cyclohexanedione graminicides appears to be primarily due to the inability of these compounds to inhibit acetyl coenzyme A carboxylase in dicotyledonous crops (Chapter 8). Monocot-dicot differences are also implied in the mode of action of auxin-type herbicides. In this case, it is speculated that the monocotyledonous auxin receptor is not accessible to the phenoxyalkanoic acids (Chapter 7).

Finally, toxic species may be inactivated before phytotoxicity is observed (Figure 2.6D). An example is the enhanced activity of superoxide dismutase in some grasses that enables relative tolerance to paraquat.

A final example of the importance of mode of action studies lies in an appreciation of the increasing problem of herbicide resistance. The prolonged use of persistent and potent herbicides with a common target site is a certain recipe for the eventual development of resistant weeds. Only by herbicide, and preferably crop, rotation can this problem be overcome. Thus, the use of herbicides with different target sites will lessen the selection pressures that favour resistance.

The most successful herbicides with negligible mammalian toxicity are those which inhibit, often selectively, metabolic processes that are unique to plants. These are principally photosynthesis, the action of plant growth regulators and the biosyntheses of pigments, lipids, and amino acids. These target processes will be considered in separate chapters of this book.

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