Interaction of Chloroplasts with Inhibitors

Several effects on pea (Pisum sativun L. var Onwards) choroplasts of a new diphenylether herbicide, fomesafen (5-12-chloro4-trifluoromethylphenoxyl-N-methanesulfonyl-2-nitrobenzamide) have been compared with those of a herbicide of related structure, nitrofluorfen (2-chloro-1-14-nitrophenoxyj4jtrlfluoromethyllbenzene). Although both compounds produce the same liht-dependent symptoms of desiccation and chlorosis indicative of a common pnmary mechanism of action, this study is concerned with a more broadly based investigation of different effects on the electron transport system. Comparsons have also been made with other compounds interacting with the chloroplast. Unlike nitrofluorfen, fomesafen has little effect as an inhibitor of electron flow or energy transfer. Both compounds have the ability to stimulate superoxide production through a functional electron transport system, and this Involves specificafly thep-nitro substituent. The stimulation, which Is not lkely to be an essential part of the prnmary herbicidal effect, is diminished under conditions that remove the coupling factor. Evidence suggests that both dipbenylethers may be able to bind to the coupling factor, and kinetic shtdis reveal this for dibromothymoquinone as well. Such a binding site might be an important feature in allowing the primary effect of the diphenylether herbicides to be expressed. Diphenylether herbicides require light for their action (7, 14) and this has focused attention on the chloroplast for the elucidation of specific sites of attack. The ability to inhibit electron transport has been established for a wide range of diphenylethers (15, 30), and Moreland et al. (15) have proposed that the main site of action is associated with PSII. Bugg et al. (3) studying nitrofluorfen' and HOE 29152 have attempted to characterize the site in more detail and found that the point of inhibition lies between the two photosystems in the plastoquinone-Cytf region. This site is similar to that inhibited by dibromothymoquinone (DBMIB) ' Abbreviations: nitrofluorfen, 2-chloro-1-(4-nitrophenoxy)-4-(trifluoromethyl)benzene; HOE 29152, methyl-2-(4-[4-trifluoromethoxy]phenoxy)propanoate; PQ, plastoquinone; DBMIB or dibromothymoquinone, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; DNP-INT, 2,4-dinitrophenylether of iodonitrothymol; acifluorfen, 5-(2-chloro-4-[trifluoromethyl]phenoxy)-2-nitrobenzoate; acifluorfen-methyl, methyl 5-(2-chloro4-[trifluoro-methyllphenoxy)-2-nitrobenzoate; fomesafen, 5-(2-chloro4trifluoromethylphenoxy)-N-methanesulfonyl-2-nitrobenzamide; TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine; 160, 50% inhibiting dose; MV, methyl viologen (paraquat); nitrofen, 2,4-dichlorophenyl-p-nitrophenyl ether, CF1, coupling factor 1; FeCN, potassium ferricyanide; oxyfluorfen, 2chloro-1-(3-ethoxy-nitrophenoxy)-4-(trifluoromethyl)benzene. and DNP-INT (29), and more specifically the inhibition involves an interaction with the Cyt b6-fcomplex close to the Rieske ironsulfur center (13). Draber et al (4) examined a number ofdiphenyl ethers for their ability to produce an inhibition pattern like DBMIB but some of these act at the same site as diuron (DCMU). Lambert et al. (10) studying the mode of action of nitrofen find that the inhibition of electron transport in spinach chloroplasts is much greater than in chloroplasts from the microalga Bumilleriopsisfliformis. There is nevertheless severe inhibition of both cyclic and noncycic photophosphorylation in chloroplasts from both species at 10 p, a concentration that does not affect electron transport in Bumilleriopsis, and nitrofen acts as an energy transfer inhibitor in strict competition with ADP in the photophosphorylation reaction. Sandmann et al. (22) have compared a range of diphenylether structures with their differing abilities to inhibit electron transport, ATP production, and induce chlorosis in the green microalga Scenedesmus acutus. One of the consequences reported for the action of diphenyl ether herbicides is an increase in permeability of cell membranes during illumination (16, 18, 31). The resulting tissue damage is essentially similar to that caused by paraquat rather than by inhibitors of electron transport, yet the bleaching effect is not reduced by Hill reaction inhibitors (14). Off and Hess (16) reported the need for another diphenylether (acifluorfen-methyl) to be activated by carotenoid to initiate a free radical chain reaction (reviewed in 17), but it can do so in the presence ofDCMU and DBMIB, which implies that the primary mechanism of action is not dependent on electron transport. Acifluorfen interferes with the blue-light-induced phototropic response and may act directly at the photoreceptor site (1 1). The present study uses a new diphenylether herbicide, fomesafen, to compare several effects on electron transport with the structurally related herbicide nitrofluorfen, with a view to learning more about the interactions with chloroplasts (whether herbicidal or not) which are common to this chemical group. Although the two diphenylethers examined have different physical properties, they produce the same symptoms of chlorosis and desiccation in plants indicative of a common primary mechanism of action. The more wide-ranging information about effects on electron transport has been obtained through a comparison of the in vitro actions produced by DCMU, DBMIB, and paraquat. CI CONHSO2CH3 CF C0jG NO2