Stefan Tresch

On the mode of action of the herbicides cinmethylin and 5-benzyloxymethyl-1, 2-isoxazolines: putative inhibitors of plant tyrosine aminotransferase.

BACKGROUND The mode of action of the grass herbicides cinmethylin and 5-benzyloxymethyl-1,2-isoxazolines substituted with methylthiophene (methiozolin) or pyridine (ISO1, ISO2) was investigated. RESULTS Physiological profiling using a series of biotests and metabolic profiling in treated duckweed (Lemna paucicostata L.) suggested a common mode of action for the herbicides. Symptoms of growth inhibition and photobleaching of new fronds in Lemna were accompanied with metabolite changes indicating an upregulation of shikimate and tyrosine metabolism, paralleled by decreased plastoquinone and carotenoid synthesis. Supplying Lemna with 10 µM of 4-hydroxyphenylpyruvate (4-HPP) reversed phytotoxic effects of cinmethylin and isoxazolines to a great extent, whereas the addition of L-tyrosine was ineffective. It was hypothesised that the herbicides block the conversion of tyrosine to 4-HPP, catalysed by tyrosine aminotransferase (TAT), in the prenylquinone pathway which provides plastoquinone, a cofactor of phytoene desaturase in carotenoid synthesis. Accordingly, enhanced resistance to ISO1 treatment was observed in Arabidopsis thaliana L. mutants, which overexpress the yeast prephenate dehydrogenase in plastids as a TAT bypass. In addition, the herbicides were able to inhibit TAT7 activity in vitro for the recombinant enzyme of A. thaliana. CONCLUSION The results suggest that TAT7 or another TAT isoenzyme is the putative target of the herbicides.

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 herbicides 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.

Inhibition of saturated very-long-chain fatty acid biosynthesis by mefluidide and perfluidone, selective inhibitors of 3-ketoacyl-CoA synthases

The trifluoromethanesulphonanilides mefluidide and perfluidone are used in agriculture as plant growth regulators and herbicides. Despite the fact that mefluidide and perfluidone have been investigated experimentally for decades, their mode of action is still unknown. In this study, we used a cascade approach of different methods to clarify the mode of action and target site of mefluidide and perfluidone. Physiological profiling using an array of biotests and metabolic profiling in treated plants of Lemna paucicostata suggested a common mode of action in very-long-chain fatty acid (VLCFA) synthesis similar to the known 3-ketoacyl-CoA synthase (KCS) inhibitor metazachlor. Detailed analysis of fatty acid composition in Lemna plants showed a decrease of saturated VLCFAs after treatment with mefluidide and perfluidone. To study compound effects on enzyme level, recombinant KCSs from Arabidopsis thaliana were expressed in Saccharomyces cerevisiae. Enzyme activities of seven KCS proteins from 17 tested were characterized by their fatty acid substrate and product spectrum. For the KCS CER6, the VLCFA product spectrum in vivo, which consists of tetracosanoic acid, hexacosanoic acid and octacosanoic acid, is reported here for the first time. Similar to metazachlor, mefluidide and perfluidone were able to inhibit KCS1, CER6 and CER60 enzyme activities in vivo. FAE1 and KCS2 were inhibited by mefluidide only slightly, whereas metazachlor and perfluidone were strong inhibitors of these enzymes with IC(50) values in μM range. This suggests that KCS enzymes in VLCFA synthesis are the primary herbicide target of mefluidide and perfluidone.

Quinclorac does not inhibit cellulose (cell wall) biosynthesis in sensitive barnyard grass and maize roots

The effect of the auxin herbicide quinclorac on cellulose, callose, and (1→3),(1→4)β-glucan deposition in newly produced cell walls of meristematic root tip cells was examined in maize and barnyard grass. Particularly, the developing cell plate of dividing cells was investigated as a site of de novo cellulose biosynthesis. A cellulose-binding domain of a bacterial cellulase and monoclonal antibodies against the hemicellulose constituents were used for specific labelling in fluorescence microscopic examination. Root-treatment of plants with 100 μM quinclorac in maize and 10 μM quinclorac in barnyard grass decreased cell division activity in root tips and root elongation. Quinclorac did not induce the swelling of root tips into a club shape and a glassy appearance of tissue, which are typical symptoms for the action of cellulose biosynthesis inhibitors such as dichlobenil. During 24 h of treatment, no effects of quinclorac on cellulose deposition at the cell plates and parental walls of meristematic root cells were found. In contrast, 10 μM dichlobenil inhibited cellulose deposition in cell plate formation within 4 h of treatment. Concerning the hemicellulose constituents, increased staining for callose in areas of cell plates and parental cell walls was observed 24 h after treatment with 10 μM quinclorac. Concomitantly, (1→3),(1→4)β-glucan deposition in cell walls decreased. The latter may be an indirect effect of quinclorac through a stimulated production of cyanide from ethylene biosynthesis. In contrast with previous reports, no evidence that quinclorac, directly or indirectly inhibits cellulose biosynthesis in roots of susceptible grasses was found.

Triaziflam and Diaminotriazine derivatives affect enantioselectively multiple herbicide target sites.

Enantiomers of triaziflam and structurally related diaminotriazines were synthesized and their herbicidal mode of action was investigated. The compounds caused light and dark-dependent effects in multiple test systems including heterotrophic cleaver and photoautotrophic algal cell suspensions, the Hill reaction of isolated thylakoids and germinating cress seeds. Dose response experiments revealed that the (S)-enantiomers of the compounds preferentially inhibited photosystem II electron transport (PET) and algae growth with efficacies similar to that of the herbicide atrazine. In contrast, the (R)-enantiomers of the diaminotriazines were up to 100 times more potent inhibitors of growth in cleaver cell suspensions and cress seedlings in the dark than the (S)-enantiomers. The most active compound, the (R)-enantiomer of triaziflam, inhibited shoot and root elongation of cress and maize seedlings at concentrations below 1 μm. The meristematic root tips swelled into a club shape which is typical for the action of mitotic disrupter herbicides and cellulose biosynthesis inhibitors. Microscopic examination using histochemical techniques revealed that triaziflam (R)-enantiomer blocks cell division in maize root tips 4 h after treatment. The chromosomes proceeded to a condensed state of prometaphase but were unable to progress further in the mitotic cycle. Disruption of mitosis was accompanied by a loss of spindle and phragmoplast micotubule arrays. Concomitantly, cortical microtubules decreased which could lead to iso-diametric cell growth and consequently to root swelling. In addition, a decline in cellulose deposition in cell walls was found 24 h after treatment. Compared to the (R)-form, triaziflam (S)-enantiomer was clearly less active. The results suggest that triaziflam and related diaminotriazines affect enantioselectively multiple sites of action which include PET inhibitory activity, mitotic disruption by inhibiting microtubule formation and inhibition of cellulose synthesis

The herbicide flamprop-M-methyl has a new antimicrotubule mechanism of action

BACKGROUND The herbicidal mode of action of flamprop-M-methyl [methyl N-benzoyl-N-(3-chloro-4-fluorophenyl)-D-alaninate] was investigated. RESULTS For initial characterization, a series of bioassays was used, which indicated a mode of action similar to that of mitotic disrupter herbicides. Cytochemical fluorescence studies, which included monoclonal antibodies against polymerized tubulin, were applied to elucidate effects on mitosis and microtubule assembly in maize roots. When seedlings were root treated with 50 microM of flamprop-M-methyl, cell division activity in meristematic root tip cells ceased within 4 h. The compound severely disturbed the orientation of spindle and phragmoblast microtubules, leading to defective spindle and phragmoblast structures. Cortical microtubules were only slightly affected. In late anaphase and early telophase cells, phragmoblast microtubules were disorganized in multiple arrays that hampered regular cell plate deposition in cytokinesis. Microtubules of the spindle apparatus were found attached to chromosomal kinetochores, but did not show regular organization associated with a zone of microtubule-organizing centres at the opposite ends of the cell. On account of this loss of spindle organization, chromosomes remained in a condensed state of prometaphase or metaphase. Unlike known microtubule disrupter herbicides, flamprop-M-methyl and its biologically active metabolite flamprop did not inhibit soybean tubulin polymerization to microtubules in vitro at 50 microM. In contrast, soybean plants responded sensitively to the compounds. CONCLUSION The results indicate that flamprop-M-methyl is a mitotic disrupter herbicide with a new antimicrotubule mechanism of action that affects orientation of spindle and phragmoblast microtubules, possibly by minus-end microtubule disassembly.

A herbicide structure-activity analysis of the antimalarial lead compound MMV007978 against Arabidopsis thaliana : Structure-activity analysis of an antimalarial lead compound against Arabidopsis thaliana

BACKGROUND To fight herbicide-resistant weeds, new herbicides are needed; particularly ones with new modes of action. Building on the revelation that many antimalarial drugs are herbicidal, here we focus on the Medicines for Malaria Venture antimalarial lead compound MMV007978 that has herbicidal activity against the model plant Arabidopsis thaliana. RESULTS Twenty-two variations of the lead compound thiophenyl motif revealed that change was tolerated provided ring size and charge were retained. MMV007978 was active against select monocot and dicot weeds, and physiological profiling indicated that its mode of action is related to germination and cell division. Of interest is the fact that the compound has a profile that is currently not found among known herbicides. CONCLUSION We demonstrate that the antimalarial compound MMV007978 is also herbicidal and that exploiting lead compounds that are often understudied could lead to the identification of interesting herbicidal scaffolds. Further structural investigation of MMV007978 could provide improved herbicidal chemistries with a potential new mode of action.

A new herbicidal site of action: Cinmethylin binds to acyl-ACP thioesterase and inhibits plant fatty acid biosynthesis

The prevalent occurrence of herbicide resistant weeds increases the necessity for new site of action herbicides for effective control as well as to relax selection pressure on the known sites of action. As a consequence, interest increased in the unexploited molecule cinmethylin as a new solution for the control of weedy grasses in cereals. Therefore, the mechanism of action of cinmethylin was reevaluated. We applied the chemoproteomic approach cellular Target Profiling™ from Evotec to identify the cinmethylin target in Lemna paucicostata protein extracts. We found three potential targets belonging to the same protein family of fatty acid thioesterases (FAT) to bind to cinmethylin with high affinity. Binding of cinmethylin to FAT proteins from Lemna and Arabidopsis was confirmed by fluorescence-based thermal shift assay. The plastid localized enzyme FAT plays a crucial role in plant lipid biosynthesis, by mediating the release of fatty acids (FA) from its acyl carrier protein (ACP) which is necessary for FA export to the endoplasmic reticulum. GC-MS analysis of free FA composition in Lemna extracts revealed strong reduction of unsaturated C18 as well as saturated C14, and C16 FAs upon treatment with cinmethylin, indicating that FA release for subsequent lipid biosynthesis is the primary target of cinmethylin. Lipid biosynthesis is a prominent target of different herbicide classes. To assess whether FAT inhibition constitutes a new mechanism of action within this complex pathway, we compared physiological effects of cinmethylin to different ACCase and VLCFA synthesis inhibitors and identified characteristic differences in plant symptomology and free FA composition upon treatment with the three herbicide classes. Also, principal component analysis of total metabolic profiling of treated Lemna plants showed strong differences in overall metabolic changes after cinmethylin, ACCase or VLCFA inhibitor treatments. Our results identified and confirmed FAT as the cinmethylin target and validate FAT inhibition as a new site of action different from other lipid biosynthesis inhibitor classes.