ISOLATION AND CHARACTERIZATION OF A CHLAMYDOMONAS-REINHARDTII MUTANT RESISTANT TO PHOTOBLEACHING HERBICIDES

A group of highly active N-phenylimide photobleaching herbicides have been synthesized. These N-phenylimide herbicides as well as diphenyl e ther herbicides induce protoporphyrin IX accumulation and inhibit prot oporphyrinogen oxidase activity at extremely low concentrations in hig her plants. The binding of a C-14-labeled N-phenylimide herbicide S-23 121 ropynyl)oxy]phenyl]-3,4,5,6-tetrahydrophthalimide] to the solubili zed plastid fractions of greening corn seedlings is competed by the di phenyl ether herbicide acifluorfen-ethyl, but not by diuron, an inhibi tor of photosynthetic electron transport. These results indicate a sim ilar mode of action for both N-phenylimide and diphenyl ether herbicid es. In order to investigate the mechanism of photobleaching herbicides at the molecular level, a strain of Chlamydomonas reinhardtii RS-3 re sistant to N-phenylimide S-23142 propargyloxyphenyl)-3,4,5,6-tetrahydr ophthalimide] was isolated by mutagenesis with N-methyl-N'-nitro-N-nit rosoguanidine. The 90% inhibition concentration of N-phenylimide S-231 42 for growth of RS-3 was 100 times higher than that for wild type. Ma ximum accumulation of protoporphyrin IX was reached at 0.03 mum of S-2 3142 for the wild type and 3 mum for RS-3. RS-3 was resistant to oxadi azon, oxyfluorfen and acifluorfen-ethyl which had been shown to have t he same mechanism of action as N-phenylimide herbicides, but not to pa raquat, diuron or fluridone. Genetic analysis of RS-3 strain showed th at the resistance results from a dominant mutation (rs-3) in the nucle ar genome. The magnesium protoporphyrin IX synthesizing activity from 5-aminolevulinic acid in chloroplast fragments isolated from RS-3 was less sensitive to S-23142 than that from wild type (CC-407). Protoporp hyrinogen oxidase activity in Percoll(TM)-purified chloroplasts from R S-3 was also less sensitive to S-23142 than that from wild type. These results indicate that the resistance of RS-3 is specific for photoble aching herbicides, and that the mutation is related to protoporphyrino gen oxidase, the primary site of the photobleaching herbicide action.