Effects of Diclofop and Diclofop-Methyl on the Membrane Potentials of Wheat and Oat Coleoptiles

Abstract

Electrophysiological measurements were made on the mesophyll cells of wheat (Triticum aesti'um L. cv Waldron) and oat (APena satiia L. cv Garry) coleoptiles treated either with the herbicide didofop-methyl (methyl 2-(4-(2',4'-dichlorophenoxy)phenoxy)propanoate), or it's primary metabolite diclofop, (2-(4-(2',4'-dichlorophenoxy)phenoxy)-propanoic acid). Application of a 100 micromolar solution of diclofop-methyl to wheat coleoptiles had little or no effect on the membrane potential (EM) however in oat, EM slowly depolarized to the diffusion potential (ED). At pH 5.7, 100 micromolar diclofop rapidly abolished the electrogenic component of the membrane potential in both oat and wheat coleoptiles with half-times of 5 to 10 minutes and 15 to 20 minutes, respectively. The concentrations giving half-maximal depolarizations in wheat were 20 to 30 micromolar compared to 10 to 20 micromolar in oat. The depolarizing response was not due to a general increase in membrane permeability as judged from the EM'S response to changes in K+, Na', Cl-, and S042-, before and after treatment with diclofop and from its response to KCN treatment. In both plants, diclofop increased the membrane permeability to protons, makin the EM strongly dependent upon the external pH in the range of pH 5.5 to pH 8.5. The effects of diclofop can best be explained by its action as a specific proton ionophore that shuttles protons across the plasmalemma. The rapidity of the cell's response to both diclofop-methyl (15-20 minutes) and diclofop (2-5 minutes) makes the ionophoric activity a likely candidate for the earliest herbicidal event exhibited by these compounds. Diclofop-methyl (methyl 2-(4-(2',4'-dichlorophenoxy)phen-oxy)propanoate) is a selective postemergence grass herbicide that controls wild oat and other grasses in wheat (1). The reasons for its phytotoxicity and selectivity are not known for certain. The selectivity is probably due to a differential metabolism between susceptible and tolerant species (8). In both susceptible oats and resistant wheat, diclofop-methyl is rapidly hydrolized to the free acid, diclofop (24442',4'-dichlorophenoxy)phenoxy)propanoic acid. In wheat diclofop is subsequently metabolized by aryl hydroxylation and conjugation to form an acidic, aryl-glucoside; in oat, diclofop is metabolized to a neutral glucosyl ester (7, 14, 21, 23). The reason for the tolerance of wheat to the herbicide is attributed to the metabolism and modification of the ring structure of diclofop as compared to ester formation in oat. It is not known for certain whether the toxic form of the herbicide is diclofop-methyl, diclofop, or both. The reason for the phytotoxicity and the mechanism of action ' Present address: Rohm and Haas Co., Research Laboratory, 727 Norristown Rd., Springhouse, PA 19477. ofdiclofop-methyl is less well understood than is the metabolism. In susceptible plants such as oat, diclofop-methyl induced an inhibition of leaf, stem, and root elongation, chlorosis of leaves (11), and severe damage to cellular ultrastructure (2). After 92 h treated leaves contained chloroplasts with compressed grana thylakoids and indistinct envelopes while the remainder of the cytoplasm had extensive vesiculation similar to that seen in senescent cells (2). Simultaneous application of diclofop-methyl and auxic herbicides such as 2,4-D under field conditions resulted in a reduction in herbicidal activity of both diclofop-methyl and the auxic compound (6). Shimabukuro et al. (22) have reported an antagonism of auxin-induced growth responses by diclofop-methyl in whole plants and in tissue culture. In oat but not wheat coleoptiles, IAA induced elongation and proton extrusion were inhibited 1 h after treatment with diclofop-methyl (21). Crowley and Prendeville (4) reported that discs from wild oat, barley, and wheat leaves treated for 12 h with diclofop-methyl had a higher rate of leakage of electrolytes than untreated leaves, suggesting that some form of membrane damage had occurred. Cohen and Morrison (3) using mitochondria isolated from both wheat and oat found that diclofop was a more potent inhibitor of state 3 respiration than was diclofop-methyl, appeared to uncouple state 4 respiration, and enhanced the rate of passive swelling of mito-chondria in isotonic KCI medium, indicating a direct influence ofdiclofop on the permeability properties ofthe inner mitochon-drial membranes. The results from various studies suggest that the herbicidal effects of diclofop-methyl or its primary metabolite diclofop, involved an increase in plasmalemma and mitochondrial membrane permeability, antagonism ofauxin mediated processes and extensive disruption of the cytoplasm. However, all of these studies used relatively long treatment times, and almost certainly involved secondary effects, or they used isolated organelles. To determine the initial, primary effects, which may be the common link to all the results discussed above, measurements of cell membrane electrical potentials were used as indicators of rapid changes in membrane activities. MATERIAILS AND METHODS To study the mode of action of diclofop-methyl and diclofop, peeled coleoptiles of oat (Avena sativa L. cv Garry), a susceptible plant, and wheat (Triticum aestivum L. cv Waldron), a resistant plant were used. Seeds of each were surface sterilized for 5 min in 1% sodium hypochlorite, rinsed for 1 h in running tap water, then were placed in paper rag dolls held upright in tap water and germinated in an incubator as described previously (20). Because the oat and wheat seeds germinated at different rates, uniform coleoptiles 25 to 30 mm in length were obtained by germinating oat seeds for 3 d and wheat seeds for 4 d at 26°C. Seeds were exposed to 4 h of red light/d to inhibit the elongation of the oat mesocotyls. 188