Probing mode of action in plant cell cycle by the herbicide endothall, a protein phosphatase inhibitor

Abstract

The mode of action of endothall, an herbicide which was reported to inhibit plant protein phosphatases 1 (PP1) and 2A (PP2A), was investigated. For initial characterization, a series of bioassays was used for comprehensive physiological profiling of endothall effects which suggested a phytotoxic mode of action similar to mitotic disrupter herbicides. Unlike known microtubule disrupters, endothall did not inhibit soybean tubulin polymerization in vitro. As shown in meristematic corn root tips, endothall distorted the orientation of cell division plane and microtubule spindle structures which led to cell cycle arrest in prometaphase. In tobacco BY-2 cells, malformed spindles together with prometaphase arrest of nuclei and abnormal perinuclear microtubule patterns were detected as early as 4 h of endothall treatment. These effects were also observed after treatment with other protein phosphatase inhibitors, cantharidin and okadaic acid, which phenocopied the mitotic changes described in tonneau1 ( ton1) and tonneau2 ( ton2) Arabidopsis mutants. These mutants are defective in TONNEAU2 (TON2) protein, a regulatory subunit of PP2A, which governs cell division plane and microtubule orientation. Therefore, PP2A/TON2 phosphatase complex is suggested to be an in planta molecular target of endothall. However, in BY-2 cells, additional effects of endothall, including inhibition of S-phase initiation and DNA synthesis, detected by 5-ethynyl-2′-deoxyuridine (EdU) incorporation, and condensed nuclei arrested in late mitosis were observed which were not reported in Arabidopsis ton1 and ton2 mutants. This result indicates that two additional checkpoints in cell cycle were blocked by endothall which are probably not associated with TON2-pathway inhibition. Possibly, inhibition of PP1 and/or other PP2A protein phosphatases are involved in the regulation of these cell cycle phenomena.