Tissue and cellular localization of acifluorfen-induced porphyrins in cucumber cotyledons

Photobleaching diphenyl ether herbicides such as acifluorfen {5-[2-chloro-4-(trifluoro-methyl)phenoxy]-2-nitrobenzoic acid} (AF) cause accumulation of high levels of protoporphyrin IX (PPIX) by inhibiting protoporphyrinogen oxidase. Previous ultrastructural studies indicated that the first sites of herbicidal actions are the plasmalemma, tonoplast, and plastid envelope, suggesting that the accumulated photodynamic pigments are operating primarily outside the plastid. Fluorescence microscopy was used to determine the tissue and cellular localization of porphyrins in acifluorfen-treated cucumber cotyledons. Yellow cotyledons, obtained by treatment with tentoxin, were used to minimize interference by chlorophyll fluorescence. Previous studies have demonstrated that tentoxin-treated cucumber tissues are highly sensitive to AF and accumulate high concentrations of PPIX. During 20 hr of dark incubation in 10 micromolar AF, 4-mm cotyledon discs accumulated 7 nmol of PPIX/g fresh weight, whereas control discs accumulated no detectable PPIX. Control and AF-treated discs accumulated 3 and 7 nmol of protochlorophyllide (PChlide)/g fresh weight, respectively, during the 20-hr incubation. In control tissues, PChlide fluorescence could be detected only in plastids; however, in AF-treated cells, fluorescence in plastids was observable earlier than in control cells, especially in cells around the periphery of the disc and around vascular bundles. After 20 hr, fluorescence in palisade cells became intense and much of the fluorescence appeared to be in the cytoplasm, outside the plastids. Occasional mesophyll cells were also affected, whereas most mesophyll cells appeared to be completely unaffected. These findings are consistent consistent with the hypothesis that the PPIX that accumulates due to photobleaching diphenyl ether herbicide treatment accumulates outside the plastid, thus affecting membranes that are relatively unprotected from toxic oxygen species.