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.
4‐Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the second reaction in the tyrosine catabolism and is linked to the production of cofactors plastoquinone and tocopherol in plants. This important biological role has put HPPD in the focus of current herbicide design efforts including the development of herbicide‐tolerant mutants. However, the molecular mechanisms of substrate binding and herbicide tolerance have yet to be elucidated. In this work, we performed molecular dynamics simulations and free energy calculations to characterize active site gating by the C‐terminal helix H11 in HPPD. We compared gating equilibria in Arabidopsis thaliana (At) and Zea mays (Zm) wild‐type proteins retrieving the experimentally observed preferred orientations from the simulations. We investigated the influence of substrate and product binding on the open–closed transition and discovered a ligand‐mediated conformational switch in H11 that mediates rapid substrate access followed by active site closing and efficient product release through H11 opening. We further studied H11 gating in At mutant HPPD, and found large differences with correlation to experimentally measured herbicide tolerance. The computational findings were then used to design a new At mutant HPPD protein that showed increased tolerance to six commercially available HPPD inhibitors in biochemical in vitro experiments. Our results underline the importance of protein flexibility and conformational transitions in substrate recognition and enzyme inhibition by herbicides.
BACKGROUND The obligatory sunflower root parasite Orobanche cumana Wallr. deprives its host of essential nutrients, resulting in a dramatic reduction in yield and biomass. A post‐emergence application with an imidazolinone herbicide on an imidazolinone‐tolerant sunflower is highly effective against O. cumana. The herbicide inhibits the enzyme acetohydroxy acid synthase and consequently, growth of the parasite is inhibited, although the sunflower survives the treatment through mutations in the target enzyme. Interestingly, field studies have shown that a combined application of an imidazolinone herbicide with prohexadione resulted in reduced emergence of O. cumana compared with the sole application of the herbicide. The aim of this study was to investigate whether prohexadione is herbicidal to O. cumana. RESULTS Prohexadione was rapidly distributed within the sunflower, reaching the roots, the site of O. cumana attack, as early as 6 h after application (HAA) on sunflower leaves. A direct impact of prohexadione on O. cumana germination was investigated and a half‐maximal inhibitory concentration (IC50) of 84 μm prohexadione was found. In addition, the inhibition of germination by prohexadione was terminal, meaning that O. cumana seeds died after prohexadione contact as soon as they were primed for germination. Additionally, excretion studies showed that a small proportion of the applied prohexadione was excreted by sunflower roots. CONCLUSION We show that prohexadione is an inhibitor of O. cumana germination and that the growth regulator is found in sunflower roots shortly after application. We hypothesize that prohexadione is excreted in sufficient amounts from the sunflower roots, therefore having a direct impact on O. cumana germination. © 2020 Society of Chemical Industry
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