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.

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The phosphoric amide herbicide amiprophos-methyl (APM) produced a concentration-dependent inhibition of taxol-induced rose microtubule polymerization in vitro. Parallel studies on taxol-induced assembly of bovine brain microtubules showed no effect of APM at a concentration ten times that required to give complete inhibition of rose microtubule assembly. The data indicate that (i) APM is a specific and potent antimicrotubule drug and (ii) APM directly poisons microtubule dynamics in plant cells, rather than indirectly depolymerizing microtubules through a previously proposed mechanism involving deregulation of intracellular calcium levels.

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