Physionomics and metabolomics-two key approaches in herbicidal mode of action discovery

BACKGROUND: For novel herbicides identified in greenhouse screens, efficient research is important to discover and chemically optimise new leads with new modes of action (MoAs). RESULTS: The metabolic and physiological response pattern to a herbicide can be viewed as the result of changes elicited in the molecular and biochemical process chain. These response patterns are diagnostic of a herbicide's MoA. At the starting point of MoA characterisation, an array of bioassays is used for comprehensive physiological profiling of herbicide effects. This physionomics approach enables discrimination between known, novel or multiple MoAs of a compound and provides a first clue to a new MoA. Metabolic profiling is performed with the use of treated Lemna paucicostata plants. After plant extraction and chromatography and mass spectrometry, changes in levels of approximately 200 identified and 300 unknown analytes are quantified. Check for known MoA assignment is performed by multivariate statistical data analyses. Distinct metabolite changes, which can direct to an affected enzymatic step, are visualised in a biochemical pathway view. Subsequent target identification includes metabolite feeding and molecular, biochemical and microscopic methods. CONCLUSION: The value of this cascade strategy is exemplified by new herbicides with MoAs in plastoquinone, auxin or very‐long‐chain fatty acid synthesis. Copyright © 2011 Society of Chemical Industry     

杂草对9类常用不同作用机制除草剂的非靶标抗性机制研究进展

除草剂的应用为农业生产带来便利, 但长期、单一使用某一种或相同机制的除草剂也引发了杂草对除草剂的抗性问题。抗性杂草种类逐渐增加, 抗性形成机制复杂, 导致农田杂草的治理难度增加。杂草对除草剂的抗性机制主要分为两种, 一种是除草剂靶标位点基因的突变或过量表达导致的靶标抗性, 另一种是杂草对除草剂吸收、转运、固存和代谢等一个或多个生理过程发生变化导致的非靶标抗性。本文综述了杂草对9类不同作用方式除草剂的非靶标抗性机制的生理、生化和分子基础的研究进展, 以期为抗性杂草综合治理提供参考。     

Status of black grass (Alopecurus myosuroides) resistance to acetyl-coenzyme A carboxylase inhibitors in France

We assessed the contributions of target site‐ and non‐target site‐based resistance to herbicides inhibiting acetyl‐coenzyme A carboxylase (ACC) in Alopecurus myosuroides (black grass). A total of 243 A. myosuroides populations collected across France were analysed using herbicide sensitivity bioassay (24 300 seedlings analysed) and ACC genotyping (13 188 seedlings analysed). Seedlings resistant to at least one ACC‐inhibiting herbicide were detected in 99.2% of the populations. Mutant, resistant ACC allele(s) were detected in 56.8% of the populations. Among the five resistant ACC alleles known in A. myosuroides, alleles containing an isoleucine‐to‐leucine substitution at codon 1781 were predominant (59.5% of the plants containing resistant ACC alleles). Comparison of the results from herbicide sensitivity bioassays with genotyping indicated that more than 75% of the plants resistant to ACC‐inhibiting herbicides in France would be resistant via increased herbicide metabolism. Analysis of herbicide application records suggested that in 15.9% of the populations studied, metabolism‐based resistance to ACC‐inhibiting herbicides was mostly selected for by herbicides with other modes of action. Our study revealed the importance of non‐target site‐based resistance in A. myosuroides. Using herbicides with alternative modes of action to control populations resistant to ACC‐inhibiting herbicides, the recommended management approach, may thus be jeopardised by the widespread occurrence of metabolism‐based resistance mechanisms conferring broad‐spectrum cross‐resistance.     

Why have no new herbicide modes of action appeared in recent years?

Herbicides with new modes of action are badly needed to manage the evolution of resistance of weeds to existing herbicides. Yet no major new mode of action has been introduced to the market place for about 20 years. There are probably several reasons for this. New potential products may have remained dormant owing to concerns that glyphosate-resistant (GR) crops have reduced the market for a new herbicide. The capture of a large fraction of the herbicide market by glyphosate with GR crops led to significantly diminished herbicide discovery efforts. Some of the reduced herbicide discovery research was also due to company consolidations and the availability of more generic herbicides. Another problem might be that the best herbicide molecular target sites may have already been discovered. However, target sites that are not utilized, for which there are inhibitors that are highly effective at killing plants, suggests that this is not true. Results of modern methods of target site discovery (e.g. gene knockout methods) are mostly not public, but there is no evidence of good herbicides with new target sites coming from these approaches. In summary, there are several reasons for a long dry period for new herbicide target sites; however, the relative magnitude of each is unclear. The economic stimulus to the herbicide industry caused by the evolution of herbicide-resistant weeds, especially GR weeds, may result in one or more new modes of action becoming available in the not too distant future.     

A novel genomic approach to herbicide and herbicide mode of action discovery

A herbicide with a new mode of action has not been commercialized for more than 30 years. A recent paper describes a novel genomic approach to herbicide and herbicide mode of action discovery. Analysis of a microbial gene cluster revealed that it encodes genes for both the biosynthetic pathway for production of the sesquiterpene aspterric acid and an aspterric acid‐resistant form of dihydroxy acid dehydratase (DHAD), its target enzyme. Aspterric acid is weak compared with commercial synthetic herbicides, and whether DHAD is a good herbicide target is unclear from this study. Nevertheless, this genomic approach provides a novel strategy for the discovery of herbicides with new modes action. © 2018 Society of Chemical Industry     

Rationale for a natural products approach to herbicide discovery

Weeds continue to evolve resistance to all the known modes of herbicidal action, but no herbicide with a new target site has been commercialized in nearly 20 years. The so-called 'new chemistries' are simply molecules belonging to new chemical classes that have the same mechanisms of action as older herbicides (e.g. the protoporphyrinogen-oxidase-inhibiting pyrimidinedione saflufenacil or the very-long-chain fatty acid elongase targeting sulfonylisoxazoline herbicide pyroxasulfone). Therefore, the number of tools to manage weeds, and in particular those that can control herbicide-resistant weeds, is diminishing rapidly. There is an imminent need for truly innovative classes of herbicides that explore chemical spaces and interact with target sites not previously exploited by older active ingredients. This review proposes a rationale for a natural-products-centered approach to herbicide discovery that capitalizes on the structural diversity and ingenuity afforded by these biologically active compounds. The natural process of extended-throughput screening (high number of compounds tested on many potential target sites over long periods of times) that has shaped the evolution of natural products tends to generate molecules tailored to interact with specific target sites. As this review shows, there is generally little overlap between the mode of action of natural and synthetic phytotoxins, and more emphasis should be placed on applying methods that have proved beneficial to the pharmaceutical industry to solve problems in the agrochemical industry.     

The use of cytochrome P450 genes to introduce herbicide tolerance in crops: a review

Mechanisms of herbicide resistance include (1) modified target site, (2) enhanced detoxification or delayed activation, and (3) alterations in the uptake, translocation, or compartmentalization of a herbicide. The first two mechanisms have mainly been identified in plants. Herbicide resistance genes were isolated for several herbicides of different modes of action. Genes that coded for herbicide target or detoxification enzymes were transferred into crop plants. The transgenic plants expressing these genes were tolerant of the active ingredients of herbicides. Before commercialization, the transgenic plants were tested in the field for risk assessment. In the case of crops with herbicide detoxification enzymes, including cytochrome-P450-species-metabolizing xenobiotics, the substrate specificity of the enzymes as well as the toxicological properties of the herbicide metabolites and the pattern of secondary metabolites in plants must be evaluated. © 1999 Society of Chemical Industry     

除草剂安全剂作用机制研究进展

除草剂安全剂在不影响除草剂对靶标杂草活性的前提下可选择性地保护作物免受除草剂的伤害。本文通过讨论安全剂对植物体内除草剂各种生理生化过程的影响,阐述了安全剂作用机制的研究。     

Flufenacet herbicide treatment phenocopies the fiddlehead mutant in Arabidopsis thaliana

In order to study the mode of action of herbicides we conducted a pilot study analysing phenotype and gene expression of flufenacet- and benfuresate-treated Arabidopsis thaliana (L) Heynhoe plants. Treatments with either herbicide caused phenocopies of the known Arabidopsis mutant fiddlehead, displaying fused organs and the typical fiddlehead-like inflorescence. Herbicide treatments of other plant species, including monocots, also gave rise to analogous organ fusions, indicating the presence of the target in a broad range of plants. Furthermore, many other herbicides with a proposed similar mode of action, eg chloroacetanilides, produced comparable fusion phenotypes in plants. The fiddlehead gene encodes a putative very-long-chain fatty acid elongase (VLCFAE), which corroborates earlier biochemical results pointing to the inhibition of VLCFA synthesis as mode of action of flufenacet. Gene expression profiles of herbicide-treated plants using the first 8247 gene Arabidopsis gene array of Affymetrix provided additional clues in support of inhibition of VLCFA synthesis. We discuss fiddlehead-like elongases as plant specific targets for flufenacet and many other herbicides.     

Physiological activity spectra of existing graminicides and the new herbicide 2-(2-benzothiazolyl-oxy)-N-methyl-N-phenylacetamide (mefenacet)

inhibitory activities of existing graminicides on root regeneration from monocotyledonous (oat) and dicotyledonous (soybean) plant cuttings in the light, in the dark and on algal growth were compared with the respective inhibitory activities of the new herbicide 2-(2-benzothiazo-lyl-oxy)-N-methyl-N-phenylacetamide (mefenacet). The mefenacet activity spectrum resembled that of the α-chloroacetamide herbicides. Herbicide groups of other structure-activity can be distinguished by their distinct activity spectrum. The mono-oxygenase inhibitors piperonyl but-oxide (PBO) and 1-aminobenzotriazole (ABT) were found to antagonize the inhibitory activities of herbicides from the thiolcarbamate, α-chloroacetamide, and oxyacetic acid amide structure groups in the oat rooting and leaf growth tests. The critical evaluation of the presently available information on graminicide and safener mode of action suggests the concept that lipid biosynthesis on the physiological level and mono-oxygenase type enzymes on the biochemical level may hold the target sites for many of the graminicides and safeners discussed.     

Recent developments in auxin biology and new opportunities for auxinic herbicide research

Auxinic herbicides mimic the effects of natural auxin. However, in spite of decades of research, the site(s) of action of auxinic herbicides has remained unknown and many physiological aspects of their function are unclear. Recent advances in auxin biology provide new opportunities for research into the mode of action of auxinic herbicides. Of considerable interest is the discovery of auxin receptors (TIR1 and possibly ABP1) that may lead to the discovery of auxinic herbicide site(s) of action. Knowledge of auxin-conjugating enzymes and auxin signal transduction components may shed new light on herbicide activity, selectivity in dicots, and mechanisms leading to phytotoxicity in sensitive plants. Analysis of genes induced in response to auxin may provide a novel approach for detection of off-target herbicide injury in crops. For example, the auxin-responsive gene GH3 is highly and specifically induced in response to auxinic herbicides in soybean, and may offer a novel method for diagnosing auxinic herbicide injury. Advances in our understanding of auxin biology will provide many new avenues and opportunities for auxinic herbicide research in the future.     

Action mechanisms of acetolactate synthase-inhibiting herbicides

Herbicides that target the acetolactate synthase (ALS) are among the most widely used weed control chemicals since their introduction into the marketplace in the early 1980s, including five classes (sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinylthio (or oxy)-benzoates and sulfonylamino-carbonyltriazolinones). The mechanism researches have progressed unprecedentedly in the last two decades. Primary mode of action of the ALS-inhibiting herbicides that interfere with the activity of ALS enzyme seems no longer in doubt. Three lines of investigation from physiology, genetics, molecular and chemical structure aspects came together to prove that ALS is the site of action. Researches on the effects of branched chain amino acids (BCAAs) synthesis or protein metabolism caused by ALS-inhibiting herbicide elicit lots of disputations. Besides these two main works, other secondary effects of ALS inhibition, such as buildup of 2-ketobutyrate (α-ketobutyrate or 2-KB) or 2-aminobutyrate (2-AB, the transamination product of 2-KB), depletion of intermediates of the pathway for some critical processes, disruption of photosynthesis transport and respiration system etc., have also been implicated in the mechanism of plant death. However, there are still some disputations and doubts on the precise mechanisms that need further probing into. Further more, as many ALS-inhibiting herbicides and their derivatives are chiral with one or even more enantiomers, which may behave quite differently in biochemical processes, the effects and the environmental fate of chiral herbicides need to be investigated stereospecifically. By this, we can have a better understanding about the herbicides and avoid unnecessary pollution load.     

Cross-resistance of Bidens subalternans to acetolactate synthase inhibitors in Brazil

Weeds resistant (R) to herbicides are widespread worldwide. Bidens subalternans is one of the most troublesome weeds in conventional soyabean fields in Brazil, and in a crop rotation system of cotton/soyabean and maize/soyabean some populations had evolved resistance to acetolactate synthase (ALS)-inhibiting herbicides. Bidens subalternans plants suspected of resistance were observed in soyabean fields where the main ALS-inhibiting herbicide sprayed is chlorimuron-ethyl. To confirm and characterise the resistance of B. subalternans to ALS inhibitors, whole-plant bioassays were conducted in 2006 and 2008. ALS in vivo enzyme bioassays were also conducted in 2007. In both bioassays, the R biotype showed cross-resistance to four chemical families of ALS-inhibiting herbicides. According to whole-plant level tests the R biotype showed 498-, 797-, 726- and >877-fold resistance to chlorimuron-ethyl, imazethapyr, cloransulam-methyl and pyrithiobac-sodium herbicides respectively. The R biotype was also 17-, 166-, 436- and 516-fold R to chlorimuron-ethyl, imazethapyr, cloransulam-methyl and pyrithiobac-sodium herbicides, respectively, based on the enzyme assay. Therefore, the herbicide-R B. subalternans can no longer be controlled by any ALS-inhibitor herbicides. Integrated control methods involving alternative herbicide with different modes of action are needed, to avoid yield losses in conventional soyabean fields in Brazil that are infested by ALS-R B. subalternans populations.     

除草剂安全剂对作物细胞色素P450及其他酶活性和水平的影响

综述了除草剂安全剂对作物中参与除草剂解毒作用的酶以及作为除草剂作用靶标位点酶水平与活性的影响。安全剂能增强细胞色素P450酶系统活性,诱导P450在除草剂降解中的作用;增加作物体内谷胱甘肽的含量,从而促进除草剂与谷胱甘肽的轭合而发挥解毒作用;降低由于除草剂对乙酰乳酸合成酶的抑制作用而引起的植物毒性等。     

Herbicide bioassay

One of the most commonly used techniques to assess the efficacy of herbicides is to apply to the principle of bioassays. A bioassay is defined as an experiment for estimating the potency of a herbicide by analysis of the reaction that follows its application to living organisms. The analysis of variance is central to most applications of statistical methods in the analysis of experiments. This is true for bioassay, but perhaps the fundamental importance of regression and related concepts is here particularly apparent. The purpose of this presentation is to quantify herbicidal effects of applying non-linear regression models to herbicide bioassays, and to demonstrate how some general hypotheses about the mode of action of the assayed herbicides can be incorporated into the regression models. The validity of herbicide bioassay data is discussed in view of the general principles used in bioassay in other biological sciences.     

具有除草活性植物病原真菌毒素的作用模式

作用模式得到明确的具有除草活性的植物病原真菌毒素为数不多,主要是危害农作物的病原真菌产生的植物毒素,目前只有少数几个具有除草活性的杂草病原真菌植物毒素的作用模式得到鉴定。而近些年来,新获得的绝大多数具有除草活性的杂草病原真菌植物毒素的作用模式尚未得到鉴定。在作用模式已经明确的具有除草活性的植物病原真菌毒素中,除了极个别与有些化学合成除草剂共享相同作用模式,绝大多数具有与现有化学合成除草剂完全不同的作用模式和独特的分子靶标位点,很有希望开发成为新型除草剂。本文重点介绍了目前已经得到鉴定的具有除草活性的植物病原真菌毒素的作用模式。     

Sulfur assimilation in plants and weed control: Potential targets for novel herbicides and action sites of certain safeners

Sulfur is an indispensable element for plants. It is found in sulfur-containing amino acids, cysteine and methionine, and in various other important biochemical components and processes. Inhibitors of sulfur assimilation, or cysteine and methionine synthesis, could be potential herbicides. In the present paper, the sulfur assimilation pathway in plants is described, followed by the introduction of several compounds (inhibitors and safeners) acting on this pathway. Uptake of inorganic sulfate through the roots is the first step of sulfur assimilation in plants. Sulfate is reduced mainly in chloroplasts to sulfide by a multistep process, and sulfide is then incorporated into cysteine. Cysteine is converted to cystathionine, homocysteine and methionine. Cysteine is incorporated into glutathione (GSH) by γ-glutamylcysteine synthetase and GSH synthetase. Three enzymes involved in cysteine and methionine biosynthesis, cysteine synthase, cystathionine γ-synthase and cystathionine β-lyase, have been investigated as target sites for herbicides. Several inhibitors of these enzymes (e.g. rhizobitoxine and propargylglycine) were also phytotoxic, suggesting that the synthetic pathway of sulfur-containing amino acids could be a new target site for herbicides. Some safeners for herbicides were found to act on the sulfur assimilation pathway and on GSH synthesis to increase GSH, which can be involved in herbicide metabolism and detoxification. Several safeners elevate GSH levels by increasing the activities of enzymes involved in sulfur assimilation and GSH synthesis. Further studies on plant sulfur metabolism may lead to the discovery of new herbicides and to the comprehensive understanding of the mode of action of safeners.     

The toxicity of herbicides to non-target aquatic plants and algae: assessment of predictive factors and hazard

Widely used herbicides sometimes inadvertently contaminate surface waters. In this study we evaluate the toxicity of herbicides to aquatic plants and algae and relate it to environmental herbicide concentrations and exposure scenarios, herbicide formulation and mode of action. This was done experimentally for ten herbicides, using the aquatic macrophyte Lemna minor L. and the green alga Pseudokirchneriella subcapitata (Korshikov) Hindak, supplemented with a database study comprising algae toxicity data for 146 herbicides. The laboratory study showed that herbicide formulations in general did not enhance herbicide efficacy in the aquatic environment. The Roundup formulation of glyphosate proved to be the only exception, decreasing the EC(50) of the technical product for both L. minor and P. subcapitata approximately fourfold. Comparison of the sensitivity of L. minor and P. subcapitata revealed up to 1000-fold higher sensitivity of L. minor for the herbicides categorized as weak acids (pK(a) < 5), emphasizing the importance of higher plants in hazard assessment. Database analyses showed that no herbicide group, categorized by site of action, was significantly more toxic than another. Synthetic auxins were the exception as they are virtually non-toxic to unicellular algae. There was no strong correlation between toxicity to algae and K(ow) of the herbicides, not even within groups having the same site of action. Evaluating all data, few herbicides were toxic at concentrations below 1 microg l(-1), which is the 99.9th percentile of the herbicide concentrations measured in the Danish surveillance programme. Joint action of several herbicides cannot however be excluded.