Enzyme activity of protoporphyrinogen-IX oxidase (Protox) prepared from 34 kinds of plant sources, containing 18 monocotyledons and 16 dicotyledons was evaluated. Protox(es) originated in monocotyledonous plants such as Zea mays cv. Anjou and DK212, Lolium perenne and Poa annua exhibited high enzyme activity, and Protox(es) in dicotyledonous plants such as Agrostemma githago and Arabidopsis thaliana showed a little higher enzyme activity than that of the above monocotyledonous plants. Highly active Protox(es) obtained from Zea mays cv. Anjou, Lolium perenne, Agrostemma githago and Arabidopsis thaliana were selected for the Protox inhibitory assay of peroxidizing herbicides. Echinochloa utilis was also selected for control. Protox inhibition by six peroxidizing herbicides such as oxyfluorfen, chlorophthalim, BW-91, pyraflufen-ethyl, DLH-1777 and LS-82556 was investigated using highly active Protox from the above monocotyledons and dicotyledons. As a result, six peroxidizing herbicides exhibited high inhibitory activity to Protox. Pyraflufen-ethyl showed the highest inhibition. The order of Protox inhibitory activity was pyraflufen-ethyl>oxyfluorfen>BW-91>chlorophthalim> DLH-1777>LS-82556, in experiment using four Protox(es) such as Zea mays cv. Anjou, Echinochloa utilis, Agrostemma githago and Arabidopsis thaliana except Lolium perenne. Oxyfluorfen exhibited the highest inhibitory activity to Protox of Lolium perenne. On the other hand, DLH-1777 showed a little less activity to Protox of Zea mays than that of other four weeds. DLH-1777 may become a model compound to find out new selective herbicides. Protox from four weeds will be used for peroxidizing herbicides assay, since the Protox showed high sensitivity to structually different peroxidizers.

Protoporphyrinogen-IXoxidase inhibition and peroxidizing phytotoxicity were determined using a series of fluorinated and non-fluorinated alkyl 2-chloro-5-(3, 4, 5, 6-tetrahydrophthalimido)benzoates and their parent benzoic acid derivatives. Protoporphyrinogen-IX oxidase from corn etioplasts was extracted, and ethane formation by intact cells of the green algae Scenedesmus acutus was measured. The benzoates with a fluorine atom exhibited stronger peroxidizing phytotoxicity (higher ethane formation) than the benzoates having no fluorine atom although protoporphyrinogen-IX oxidase inhibition by the fluorinated analogues was found to be weaker or almost equal to the non-fluorinated analogues. These data strengthen and generalize our previous findings with cyclic imides having an N-(4-chloro-2-fluoro-5-carboxyphenyl) group. The fluorinated analogues, which are not necessarily stronger protoporphyrinogen-IX oxidase inhibitors, exhibit stronger peroxidizing phytotoxicity with intact plant cells than those without a fluorine atom.

Peroxidizing herbicides have a common mode of action, i. e. inhibition of chlorophyll biosynthesis. A molecular target of these herbicides is protoporphyrinogen-IX oxidase (Protox). Corn is widely used for Protox inhibitory assays by peroxidizing herbicides because the plant enzyme has high and stable activity. The Protox isolated from one corn variety, Anjou, has been used for the assay in our laboratory and several others. This variety can no longer be used however, because of its reduced tolerance against corn diseases. Therefore, we collected twelve corn varieties and compared their Protox activities and sensitivities against peroxidizing herbicides. Among the tested varieties, DK212MF exhibited the highest activity. This variety has approximately two times higher activity than Anjou. Tolerance of Anjou and DK212MF to peroxidizing herbicides was evaluated by a pot test, because the varieties have quite different Protox activities. DK212MF was less sensitive to the peroxidizing herbicides oxyfluorfen and chlorophthalim than Anjou. Furthermore, DK212MF accumulated less Proto-IX than Anjou, which was believed to be one of the causes of its lower sensitivity in vivo to the peroxidizing herbicides.