20.2.1  Target site resistance

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Genetic resistance to a herbicide is achieved if changes in a gene encode a structural change in its gene product (enzyme), such that the herbicide no longer binds in an inhibitory manner. Of course, any such structural change to the enzyme must not also inactivate normal function of the enzyme. Therefore, only a few mutations confer resistance. None the less, ‘modified target site’ resistance is common in huge weed populations exposed to herbicides. Three major cases of target site resistance are outlined below.

(a)  Photosystem II target site resistance

Widely used triazine herbicides inhibit photosynthesis at photosystem II (PSII). PSII is one of the pigment–protein complexes embedded within the thylakoid membrane system of chloroplasts essential to the capture of light energy (Chapter 1). Triazine herbicides bind to the QB niche within the D1 protein of PSII, effectively blocking electron transport from PSII and thereby inhibiting photosynthesis. There is extensive resistance in many weed species to triazine herbicides around the world. With a few exceptions, triazine resistance is target-site based, entailing a single amino acid change in the D1 protein of PSII. The substitution of serine at position 264 with glycine dramatically reduces binding of triazine herbicides to the D1 protein and renders these plants resistant (Figure 20.3). This mutation endows triazine resistance without severe inhibition of QB binding to D1 protein, leaving enough photosynthetic capacity for survival. Only this mutation is observed in triazine-resistant weeds, indicating that other changes to the QB niche vastly reduce fitness.


Figure 20.3 Schematic diagram showing the interaction of atrazine with D1 protein of PSII. (a) Atrazine interacts with serine at position 264, binding tightly to the D1 protein. In the herbicide-resistant enzyme (b), serine 264 has been replaced by glycine, which cannot interact with atrazine. (c) Inhibition of PSII activity by atrazine for thylakoids isolated from susceptible and resistant biotypes of Chenopodium album. (Based on Fuerst et al. 1986 and Fuerst and Norman 1991)


(b)  Acetolactate synthase (ALS) target site resistance

ALS, also known as acetohydroxyacid synthase, catalyses the first step in biosynthesis of the branched-chain amino acids leucine, valine and isoleucine. This enzyme catalyses two reactions: (1) condensation of two molecules of pyruvate to form α-acetolactate and (2) condensation of pyruvate and α-ketobutyrate to form α-acetohydroxybutyrate. Several commercial herbicide groups are widely used as potent inhibitors of ALS. Resistance to an ALS-inhibiting herbicide was first detected in Australia in L. rigidum in 1984, shortly after the release of chlorsulfuron, the first of these herbicides. Since then, hundreds of L. rigidum populations have become resistant to ALS-inhibiting herbicides as well as populations of 13 other weed species including Brassica tournefortii, Raphanus raphanistrum and Echium plantagineum. Resistance has also appeared in a range of weed species in North America and elsewhere in the world. Resistance to ALS-inhibiting herbicides is often endowed by a resistant form of the target enzyme, ALS. Resistant ALS enzymes are present in many populations of L. rigidum and all ALS-resistant dicotyledonous weed populations examined to date. However, the response of these resistant enzymes to different ALS-inhibiting herbicides is not the same. Some biotypes contain an enzyme that is highly resistant to all three classes of ALS-inhibiting herbicides, whereas others contain an enzyme that is susceptible to one or more classes. These differences between resistant enzymes suggest there are different mutations within the ALS-encoding gene that render the enzyme resistant. Molecular studies with the gene confirm this. The ALS-encoding gene contains two highly conserved regions, Domains A and B, where mutations endowing resistance have been found (Figure 20.4). Within Domain A, substitution of a conserved proline residue with a range of other amino acids confers resistance to some but not all ALS-inhibiting herbicides. Within Domain B, substitution of leucine for a conserved tryptophan endows strong resistance to a wider range of ALS-inhibiting herbicides. Hence, several mutations in the ALS-encoding gene confer herbicide resistance without compromising enzyme function.


Figure 20.4 (a) Schematic diagram of ALS showing the herbicide-binding domain, consisting of the highly conserved amino-acids in Domains A and B. In Domains A and B of herbicide-susceptible ALS, proline is found at position 197 (Pro 197) and tryptophan at position 574 (Trp 574), respectively. The numbering sequence is that used for Arabidopsis thaliana (Mazur et al. 1987). Substitutions of alanine, arginine, glutamine, histidine, isoleucine, leucine, serine or threonine for Pro 197 endow resistance to ALS-inhibiting herbicides. Likewise, substitution of leucine for Trp 574 also endows resistance. (b) Chlorosulfuron inhibition in vitro of ALS from susceptible (Ο) and two resistant populations of Sisymbrium orientale containing substitutions of isoluecine for proline 197 (●) or leucine for tryptophan 574 (). (Based on O. Boutsalis and S.B. Powles, unpublished data)

(c)  Acetyl coenzyme A carboxylase (ACCase) target site resistance

ACCase catalyses the first step in fatty acid biosynthesis, namely carboxylation of acetyl-coenzyme A to produce malonyl-coenzyme A. ACCase is a multifunctional enzyme predominantly located in chloroplasts. There are also extra-chloroplastic isomers of ACCase which participate in synthesis of very long chain fatty acids for cutin and flavonoids. ACCase-inhibiting herbicides reduce fatty acid synthesis, most noticeably in young actively growing tissues where demand for lipids is high. Two structurally different herbicides inhibit ACCase, the aryoxyphenoxypropanoates, such as diclofop-methyl, and the cyclohexanediones, such as sethoxydim.


Table 20.1

ACCase-inhibiting herbicides are unusual in that they are active only on grass species. Indeed, the chloroplastic ACCase of dicotyledonous species is structurally different from that of grass species and is resistant to these herbicides. ACCase-inhibiting herbicides are used widely in Australia and resistance to these herbicides is now a major practical problem. Indeed, the first documented case of resistance to ACCase-inhibiting herbicides was a population of L. rigidum in Australia. Since then there have been many hundreds of cases of resistance to ACCase-inhibiting herbicides, principally in L. rigidum, but also increasingly in wild oat (Avena fatua and A. sterilis) and in other grass weeds. Resistance to ACCase herbicides is now a widespread problem throughout cropping regions of southern Australia.

Many cases of resistance to ACCase-inhibiting herbicides in L. rigidum and other resistant species are the result of a resistant target enzyme. Like ALS, resistant ACCase enzymes from different biotypes have different responses to ACCase-inhibiting herbicides (Table 20.1). Different mutations of ACCase appear to confer resistance without compromising enzyme function. It is anticipated that once ACCase-encoding genes are cloned and sequenced, amino acid changes that confer resistance will be identified.