Hideyuki Shibata

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 ether herbicides induce protoporphyrin IX accumulation and inhibit protoporphyrinogen oxidase activity at extremely low concentrations in higher plants. The binding of a 14C -labeled N -phenylimide herbicide S-23121 [N-[4-chloro- 2-fluoro-5-[(1-m ethyl-2-propynyl)oxy]phenyl]-3,4,5,6-tetrahydrophthalimide] to the solubilized plastid fractions of greening corn seedlings is competed by the diphenyl ether herbicide acifluorfen-ethyl, but not by diuron, an inhibitor of photosynthetic electron transport. These results indicate a similar mode of action for both N -phenylimide and diphenyl ether herbicides. In order to investigate the mechanism of photobleaching herbicides at the molecular level, a strain of Chlamydomonas reinhardtii RS-3 resistant to N -phenylimide S-23142 [N -(4-chloro- 2-fluoro-5-propargyloxyphenyl)-3,4,5,6-tetrahydrophthalimide] was isolated by mutagenesis with N -m ethyl-N′-nitro-N -nitrosoguanidine. The 90% inhibition concentration of N -phenylimide S-23142 for growth of RS-3 was 100 times higher than that for wild type. Maximum accumulation of protoporphyrin IX was reached at 0.03 μᴍ of S-23142 for the wild type and 3 μᴍ for RS-3. RS-3 was resistant to oxadiazon, oxyfluorfen and acifluorfen-ethyl which had been shown to have the same mechanism of action as N -phenylimide herbicides, but not to paraquat, diuron or fluridone. Genetic analysis of RS-3 strain showed that the resistance results from a dominant mutation ( rs-3) in the nuclear genome. The magnesium protoporphyrin IX synthesizing activity from 5-am inolevulinic acid in chloroplast fragments isolated from RS-3 was less sensitive to S-23142 than that from wild type (CC-407). Protoporphyrinogen oxidase activity in Percoll™ -purified chloroplasts from RS-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 photobleaching herbicides, and that the mutation is related to protoporphyrinogen oxidase, the primary site of the photobleaching herbicide action.