Aclonifen is a unique diphenyl ether herbicide. Despite its structural similarities to known inhibitors of the protoporphyrinogen oxidase (e.g., acifluorfen, bifenox or oxadiazon), which result in leaf necrosis, aclonifen causes a different phenotype that is described as bleaching. This is also reflected by the Herbicide Resistance Action Committee (HRAC) classification that categorizes aclonifen as an inhibitor of pigment biosynthesis with an unknown target.A comprehensive Arabidopsis thaliana RNAseq dataset comprising 49 different inhibitor treatments and covering 40 known target pathways was used to predict the aclonifen mode of action (MoA) by a random forest classifier. The classifier predicts for aclonifen a MoA within the carotenoid biosynthesis pathway similar to the reference compound norflurazon that inhibits the phytoene desaturase. Upon aclonifen treatment, the phytoene desaturation reaction is disturbed, resulting in a characteristic phytoene accumulation in vivo. However, direct enzyme inhibition by the herbicide was excluded for known herbicidal targets such as phytoene desaturase, 4-hydroxyphenylpyruvate dioxygenase and homogentisate solanesyltransferase. Eventually, the solanesyl diphosphate synthase, providing one of the two homogentisate solanesyltransferase substrate molecules, could be identified as the molecular target of aclonifen. Inhibition was confirmed using biochemical activity assays for the Arabidopsis thaliana solanesyl diphosphate synthases 1 and 2. Furthermore, a Chlamydomonas reinhardtii homolog was used for co-crystallization of the enzyme-inhibitor complex, showing that one inhibitor molecule binds at the interface between two protein monomers.Solanesyl diphosphate synthase was identified as the target of aclonifen, representing a novel mode of action for herbicides. This article is protected by copyright. All rights reserved.
