Oryzalin, a dinitroaniline herbicide, binds to plant tubulin and inhibits microtubule polymerization in vitro

Abstract

The effects of oryzalin, a dinitroaniline herbicide, on chromosome behavior and on cellular microtubules (MTs) were examined by light microscopy and immunogold staining, respectively, in endosperm cells from Haemanthus katherinae Bak. Brief treatments with 1.0·10(-8) M oryzalin reduced markedly the migration rate of anaphase chromosomes and 1.0·10(-7) M oryzalin stopped migration abruptly. Oryzalin (1.0·10(-7) M) depolymerized MTs and prevented the polymerization of new MTs at all stages of the mitotic cycle. The chromosome condensation cycle was unaffected by oryzalin. Endothelial cells from the heart of Xenopus leavis showed no chromosomal or microtubular rearrangements after oryzalin treatment. The inhibition by oryzalin of the polymerization of tubulin isolated from cultured cells of Rosa sp. cv. Paul's scarlet was examined in vitro by turbidimetry, electron microscopy and polymer sedimentation analysis. Oryzalin inhibited the rapid phase of taxol-induced polymerization of rose MTs at 24°C with an apparent inhibition constant (K i ) of 2.59·10(6) M. Shorter and fewer MTs were formed with increasing oryzalin concentrations, and maximum inhibition of taxol-induced polymerization occurred at approx. 1:1 molar ratios of oryzalin and tubulin. Oryzalin partially depolymerized taxol-stabilized rose MTs. Ligand-binding experiments with [(14)C]oryzalin demonstrated the formation of a tubulin-oryzalin complex that was time- and pH-dependent. The tubulin-oryzalin interaction (24°C, pH 7.1) had an apparent affinity constant (K app) of 1.19·10(5) M(-1). Oryzalin did not inhibit taxol-induced polymerization of bovinebrain MTs and no appreciable binding of oryzalin to brain tubulin or other proteins was detected. The results demonstrate pharmacological differences between plant and animal tubulins and indicate that the most sensitive mode of action of the dinitroaniline herbicides is the direct poisoning of MT dynamics in cells of higher plants.

Rapid and Reversible High-Affinity Binding of the Dinitroaniline Herbicide Oryzalin to Tubulin from Zea mays L

“…Although the maximum aqueous solubility of oryzalin is reported to be 7.2 ,UM (Fedtke, 1982), at concentrations substantially below this, we found that oryzalin is lost from solution by binding to glass or plastic container surfaces. Inclusion in the buffer of a low concentration of a polar solvent enhances slightly the solubility of oryzalin in aqueous solutions (Strachen and Hess, 1982;Morejohn et al, 1987a). For the current work, a11 solutions of IB contained a final concentration of 2% (v/v) DMSO.…”

“…Our previous work has shown that low micromolar concentrations of oryzalin inhibit the taxol-induced polymerization of tubulin isolated from cultured cells of rose (Rosa sp.) (Morejohn et al, 1987a). Ligand-binding experiments using the "kinetic" ligand-binding method of DEAE-cellulose disc filtration showed that oryzalin binds slowly to rose tubulin to form a TO complex with an apparent Kd = 8.4 PM (K, = 1.19 X 105 M -' ) , and maximum r of 0.14.…”

“…However, the relatively high & and low binding stoichiometry have appeared inconsistent with the observations that much lower oryzalin concentrations (2100 nM) rapidly depolymerize microtubules and inhibit anaphase chromosome migration in isolated endosperm cells from Hemanthus katherinae Bak. (Morejohn et al, 1987a). Ligand-binding and polymerization experiments were performed with a buffer containing 1 M SUC, a tubulin-stabilizing component known to prevent the normal decay of colchicine binding to tubulin (Frigon and Lee, 1972).…”

Preprophase band formation and cortical division zone establishment: RanGAP behaves differently from microtubules during their band formation

“…To modify the MT band, we employed 2 different methods using cytoskeletal drugs. The application of oryzalin, an inhibitor of plant MT polymerization, 56 to root tip cells may induce the depolymerization of MTs in a PPB. Hence, the number of MTs in PPB is expected to decrease or MTs are expected to disperse shortly after oryzalin application.…”

Effects of herbicides on growth and survival, and influence of latitude on anastomosis group diversity in Rhizoctonia solani

“…Pendimethalin and other dinitroanaline herbicides have been shown to affect microtubule (MT) formation in algae (Strachan and Hess 1983), protozoans (Chan et aL 1991), and fungi (Hyde and Hardham 1993). Dinitroanaline herbicides lead to depolymerization of existing MTs and prevent the polymerization of new ones (Morejohn et al 1987;Wacker et al 1987). Unlike pendimethalin, mycelial growth was not inhibited by imazethapyr and glyphosate.…”