Competitive Inhibition of High-Affinity Oryzalin Binding to Plant Tubulin by the Phosphoric Amide Herbicide Amiprophos-Methyl

Abstract

Amiprophos-methyl (APM), a phosphoric amide herbicide, was previously reported to inhibit the in vitro polymerization of isolated plant tubulin (L.C. Morejohn, D.E. Fosket [1984] Science 224: 874-876), yet little other biochemical information exists concerning this compound. To characterize further the mechanism of action of APM, its interactions with tubulin and microtubules purified from cultured cells of tobacco (Nicofiana tabacum cv Bright Yellow-2) were investigated. Low micromolar concentrations of APM depolymerized preformed, taxol-stabilized tobacco microtubules. Remarkably, at the lowest APM concentration examined, many short microtubules were redistributed into fewer but 2.7-fold longer microtubules without a substantial decrease in total polymer mass, a result consistent with an end-to-end annealing of microtubules with enhanced kinetic properties. Quasi-equilibrium binding measurements showed that tobacco tubulin binds ['4C]oryzalin with high affinity to produce a tubulin-oryzalin complex having a dis- Phosphoric amide herbicides such as APM (Tokunol M) and butamiphos (Cremart) were developed as preemergence herbicides and are effective on annual grasses and broadleaf weeds (Aya et al., 1975). The uses of phosphoric amides are similar to those of the dinitroaniline herbicides, including

Dinitroanilines Bind α-Tubulin to Disrupt Microtubules

“…For each ␣-tubulin conformation, we found the consensus binding site ( Figure 5A) was both the largest cluster and contained the binding conformations with the highest affinity. The binding affinity predicted for this site was 23 nM and is in agreement with experimentally determined values of 95-117 nM obtained with purified plant tubulin (Hugdahl and Morejohn, 1993;Murthy et al, 1994). The site identified by our docking simulations is located beneath the N loop and is composed of residues Arg2, Glu3, Val4, Trp21, Phe24, His28, Ile42, Asp47, Arg64, Cys65, Thr239, Arg243, and Phe244 ( Figure 5A).…”

“…Dinitroanilines also disrupt the microtubules of protozoa, including both free-living species such as Tetrahymena and protozoan parasites such as Trypanosoma spp., Leishmania spp., Entamoeba spp., Plasmodium falciparum, Cryptosporidium parvum, and Toxoplasma gondii (Chan and Fong, 1990;Chan et al, 1991;Gu et al, 1995;Edlind et al, 1996;Stokkermans et al, 1996;Armson et al, 1999; Makioka et al, 2000a,b;Traub-Cseko et al, 2001). Remarkably, the activity of dinitroanilines is restricted to plants and protozoa; these compounds are ineffective against vertebrate or fungal microtubules (Chan and Fong, 1990;Hugdahl and Morejohn, 1993;Murthy et al, 1994;Edlind et al, 1996).T. gondii is a member of the Apicomplexa, a phylum of parasites that includes several medically and agriculturally significant pathogens (Black and Boothroyd, 2000).…”

Tobacco mosaic virus infection spreads cell to cell as intact replication complexes

“…Amiprophos-methyl, a phosphoric amide herbicide, inhibits the polymerization of isolated plant tubulin in vitro (34) and the formation of microtubules in tobacco suspension cells (35,36). After injection of amiprophos-methyl (50 M solution) into the leaf tissue, we did not detect changes in the morphology or localization of VRC, and the fluorescent ring of virus infection continued to spread.…”

The POLARIS Peptide of Arabidopsis Regulates Auxin Transport and Root Growth via Effects on Ethylene Signaling

“…Furthermore, transgenic plants overexpressing the PLS open reading frame showed a more severe response to APM than did wild-type plants ( Figure 6D). Oryzalin, a second microtubule herbicide that is chemically dissimilar to APM, although more potent (Murthy et al, 1994), but which is known to interact with the same target site on tubulin (Ellis et al, 1994), also induced a less severe response in pls compared with wild-type plants ( Figure 6E). A third antimicrotubule herbicide, propyzamide, which is of a similar size but has a different mode of action than APM and oryzalin (Anthony et al, 1998), has identical inhibitory effects on root growth of wild-type, pls, and PLSoverexpresser plants ( Figures 6F and 6H).…”

“…pls seedlings show reduced responses to the microtubule inhibitors APM and oryzalin. These inhibitors are members of different chemical classes (phosphorothioamidates and dinitroanilines, respectively), but they have the same predicted mode of action, in the binding of tubulin and the subsequent inhibition of tubulin polymerization (Murthy et al, 1994;Anthony and Hussey, 1999a). This affects microtubule dynamics (by destabilizing them), leading to an inhibition of cell division and axial cell expansion.…”