Johannes Hutzler

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.

Saflufenacil (Kixor™): Biokinetic Properties and Mechanism of Selectivity of a New Protoporphyrinogen IX Oxidase Inhibiting Herbicide

Abstract Saflufenacil (Kixor™) is a new protoporphyrinogen IX oxidase (PPO) inhibiting herbicide for preplant burndown and selective PRE dicot weed control in multiple crops, including corn. The biokinetic properties and the mechanism of selectivity of saflufenacil in corn, black nightshade, and tall morningglory were investigated. After root treatment of plants at the third-leaf stage, the difference in the phytotoxic selectivity of saflufenacil in corn and the weed species has been quantified as approximately 10-fold. The plant species showed similar selectivity after foliar applications; the plant response to saflufenacil was approximately 100-fold more sensitive compared with a root application. PPO enzyme activity in vitro was inhibited by saflufenacil, a 50% inhibition lay in a concentration range from 0.2 to 2.0 nM, with no clear differences between corn and the weed species. Treatments of light-grown plants and dark-grown seedlings with [14C]saflufenacil revealed that the herbicide is rapidly absorbed by root and shoot tissue. The [14C]saflufenacil was distributed within the plant systemically by acropetal and basipetal movement. Systemic [14C]saflufenacil distribution can be explained by the weak acid character of saflufenacil and its metabolic stability in black nightshade and tall morningglory. Metabolism of [14C]saflufenacil in corn was more rapid than in the weeds. In addition, low translocation of root-absorbed [14C]saflufenacil in the corn shoot was observed. It is concluded that rapid metabolism, combined with a low root translocation, support PRE selectivity of saflufenacil in corn. Nomenclature: Butafenacil; flumioxazin; saflufenacil; black nightshade, Solanum nigrum L., SOLNI; corn, Zea mays L., ZEAMX; tall morningglory, Ipomoea purpurea (L.) Roth. PHBPU; velvetleaf, Abutilon theophrasti Medik., ABUTH.

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.

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.