The nexus between reactive oxygen species and the mechanism of action of herbicides.

Herbicides are small molecules that act by inhibiting specific molecular target sites within primary plant metabolic pathways resulting in catastrophic and lethal consequences. The stress induced by herbicides generates reactive oxygen species (ROS), but little is known about the nexus between each herbicide mode of action (MoA) and their respective ability to induce ROS formation. Indeed, some herbicides cause dramatic surges in ROS levels as part of their primary MoA, whereas other herbicides may generate some ROS as a secondary effect of the stress they imposed on plants. In this review, we discuss the types of ROS and their respective reactivity and describe their involvement for each known MoA based on the new Herbicide Resistance Action Committee classification.

Tetrahydroquinolinyl phosphinamidates and phosphonamidates enhancing tolerance towards drought stress in crops via interaction with ABA receptor proteins.

New phosphorous-containing lead structures against drought stress in crops interacting with RCAR/(PYR/PYL) receptor proteins were identified starting from in-depth SAR studies of related sulfonamide lead structures and protein docking studies. A converging 6-step synthesis via phosphinic chlorides and phosphono chloridates as key intermediates afforded envisaged tetrahydroquinolinyl phosphinamidates and phosphonamidates. Whilst tetrahydroquinolinyl phosphonamidates 13a,b exhibited low to moderate target affinities, the corresponding tetrahydroquinolinyl phosphinamidates 12a,b revealed confirmed strong affinities for RCAR/ (PYR/PYL) receptor proteins in Arabidopsis thaliana on the same level as essential plant hormone abscisic acid (ABA) combined with promising efficacy against drought stress in vivo (broad-acre crops wheat and canola).

Identifying new lead structures to enhance tolerance towards drought stress via high-throughput screening giving crops a quantum of solace.

Novel synthetic lead structures interacting with RCAR/(PYR/PYL) receptor proteins were identified based on the results of a high-throughput screening campaign of a large compound library followed by focused SAR studies of the three most promising hit clusters. Whilst indolinylmethyl sulfonamides 8y,z and phenylsulfonyl ethylenediamines 9y,z showed strong affinities for RCAR/ (PYR/PYL) receptor proteins in wheat, thiotriazolyl acetamides 7f,s exhibited promising efficacy against drought stress in vivo (wheat, corn and canola) combined with confirmed target interaction in wheat and arabidopsis thaliana. Remarkably, binding affinities of several representatives of 8 and 9 were on the same level or even better than the essential plant hormone abscisic acid (ABA).

The identification of small molecule inhibitors of the plant inositol phosphorylceramide synthase which demonstrate herbicidal activity.

Resistance to 157 different herbicides and 88% of known sites of action has been observed, with many weeds resistant to two or more modes. Coupled with tighter environmental regulation, this demonstrates the need to identify new modes of action and novel herbicides. The plant sphingolipid biosynthetic enzyme, inositol phosphorylceramide synthase (IPCS), has been identified as a novel, putative herbicide target. The non-mammalian nature of this enzyme offers the potential of discovering plant specific inhibitory compounds with minimal impact on animals and humans, perhaps leading to the development of new non-toxic herbicides. The best characterised and most highly expressed isoform of the enzyme in the model-dicot Arabidopsis, AtIPCS2, was formatted into a yeast-based assay which was then utilized to screen a proprietary library of over 11,000 compounds provided by Bayer AG. Hits from this screen were validated in a secondary in vitro enzyme assay. These studies led to the identification of a potent inhibitor that showed selectivity for AtIPCS2 over the yeast orthologue, and activity against Arabidopsis seedlings. This work highlighted the use of a yeast-based screening assay to discover herbicidal compounds and the status of the plant IPCS as a novel herbicidal target.

Stable expression and functional characterisation of the diamondback moth ryanodine receptor G4946E variant conferring resistance to diamide insecticides.

Diamides, such as flubendiamide and chlorantraniliprole, belong to a new chemical class of insecticides that act as conformation-sensitive activators of insect ryanodine receptors (RyRs). Both compounds are registered for use against lepidopteran species such as the diamondback moth, Plutella xylostella, a notorious global pest of cruciferous crops. Recently acquired resistance to diamide insecticides in this species is thought to be due to a target-site mutation conferring an amino acid substitution (G4946E), located within the trans-membrane domain of the RyR, though the exact role of this mutation has not yet been fully determined. To address this we have cloned a full-length cDNA encoding the P. xylostella RyR and established clonal Sf9 cell lines stably expressing either the wildtype RyR or the G4946E variant, in order to test the sensitivity to flubendiamide and chlorantraniliprole on the recombinant receptor. We report that the efficacy of both diamides was dramatically reduced in clonal Sf9 cells stably expressing the G4946E modified RyR, providing clear functional evidence that the G4946E RyR mutation impairs diamide insecticide binding.

Phthalic acid diamides activate ryanodine-sensitive Ca2+ release channels in insects.

Flubendiamide represents a novel chemical family of substituted phthalic acid diamides with potent insecticidal activity. So far, the molecular target and the mechanism of action were not known. Here we present for the first time evidence that phthalic acid diamides activate ryanodine-sensitive intracellular calcium release channels (ryanodine receptors, RyR) in insects. With Ca(2+) measurements, we showed that flubendiamide and related compounds induced ryanodine-sensitive cytosolic calcium transients that were independent of the extracellular calcium concentration in isolated neurons from the pest insect Heliothis virescens as well as in transfected CHO cells expressing the ryanodine receptor from Drosophila melanogaster. Binding studies on microsomal membranes from Heliothis flight muscles revealed that flubendiamide and related compounds interacted with a site distinct from the ryanodine binding site and disrupted the calcium regulation of ryanodine binding by an allosteric mechanism. This novel insecticide mode of action seems to be restricted to specific RyR subtypes because the phthalic acid diamides reported here had almost no effect on mammalian type 1 ryanodine receptors.