Four substituted pyridazinone compounds inhibited the Hill reaction and photosynthesis in barley (Hordeum vulgareL., var. Dayton C.I. 9517). These inhibitions appeared to account for the phytotoxicity of 5-amino-4-chloro-2-phenyl-3(2H)-pyridazinone (pyrazon). The pyridazinone chemicals were weaker inhibitors than 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (atrazine). Two substitutions onto the molecular structure of pyrazon result in a new experimental herbicide, 4-chloro-5-(dimethylamino)-2-(α,α,α-trifluoro-m-tolyl)-3(2H)-pyridazinone (hereinafter referred to as 6706), which retains the action mechanism of pyrazon but also has two additional biological properties. It is resistant to metabolic detoxication in plants, and it possesses a second mode of action involving interference with chloroplast development. The second action is like that expressed by 3-amino-s-triazole (amitrole) and by 3,4-dichlorobenzyl methylcarbamate (dichlormate). However, the new chemical is 100 to 1000 times more effective. The trifluoromethyl substitution on the phenyl ring and the dimethyl substitution on the amine are both required to give either of the two additional physiological properties. Analogs with only one of the two substitutions behave like pyrazon rather than like 6706.

Chloroplasts were isolated from triazine-sensitive and triazine-resistant biotypes of common groundsel (Senecio vulgaris L.), common lambsquarter (Chenopodicm album L.), and redroot pigweed (Amarathu retrojexus L.). Chloroplast lipids were extracted and analyzed for differences among sensitive and resistant biotypes. (2) and Pfister et al. (9) have recently reviewed studies which provide evidence defining the mode and site ofaction oftriazine herbicides as well as the mechanism responsible for herbicide resistance.Extensive evidence suggests that the triazine herbicides inhibit PSII; specifically, the secondary electron carrier on the reducing side of PSII (called B) has been proposed as the target site for these compounds.The developed resistance to triazines is apparently related to alterations at the target site. Arntzen et al. (2) and Pfister et al. (9) suggest that triazine resistance of both intact plants and isolated chloroplasts of common groundsel is based upon a minor modification of a protein in the PSII complex responsible for herbicide binding. This change could result in a specific loss of herbicidebinding capacity.In addition to the herbicide-binding protein, the proposed model for the PSII complex consists of light-harvesting and reaction center pigments, specific enzymatic proteins, and a chain of electron carriers localized within the chloroplast thylakoid membranes. Thus, ordered interactions ofproteins, pigments, and lipids would be required for functional activity of the complexes. The present studies compared the lipid composition of chloroplast membranes from weed biotypes differentially sensitive to triazines. The purpose was to focus on the mechanism ofherbicide resistance and, in addition, to characterize one of the major membrane components possibly influencing the functioning of the PSII complex. MATERIALS AND METHODSSeeds of common groundsel, common lambsquarters (Chenopodium album L.), and redroot pigweed (Amaranthus retroflexus L.) germinated in flats containing Jiffy Mix' (Jiffy Products of America, West Chicago, IL) in a growth chamber with a 16-h photoperiod in which day temperature was 27 ± 1 C and night temperature was 21 ± 1 C. Flats were kept moist with tap water. After two weeks, healthy seedlings were transplanted into individual pots (95 x 95 x 85 mm) and were grown in a greenhouse with an average temperature of 27 ± 1 C night. Four-week-old plants were used for isolation of chloroplasts.Isolation of Chloroplast. Chloroplasts were isolated essentially according to the procedure of Nakatani and Barber (8). Isolated chloroplasts were intact, as judged by ferricyanide reduction. Chl was determined by the method of Arnon (1).Fatty Acid Analysis. Lipids were extracted and recovered from lypholized chloroplasts, according to the procedures of Folch et aL (6). Polar lipids were separated from nonpolar lipids by the rubber membrane dialysis method of Bottcher et aL (4). Free fatty acids were separated by washing the nonpolar lipids with 25 to 30 ml of 0.05 N KOH. The pol...

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Formation of chloroplast pigments was inhibited, and free fatty acids accumulated in mustard (Brassica juncea [L.] Coss.) cotyledons and in barley (Hordeum vulgare L.) first leaves developed after treatment with 4-chloro-5-(dimethylamino)-2-(a, a, a-trifluoro-m-tolyl)-3 (2H)-pyridazinone. The inhibitor reduced the amount of fatty acids found in polar lipids (galactolipids) of barley chloroplasts and increased the amount in nonpolar lipids while having little effect on total content of bound fatty acids. The inhibition of chlorophyll formation was circumvented by D-a-tocopherol acetate, phytol, farnesol, and squalene, and by unsaturated fatty acids and their methyl esters. The protective action can be explained partially by an interaction external to the plant whereby 4-chloro-5-(dimethylamino)-2-(a, a, a-trifluoro-m-tolyl) -3 (2H)-pyridazinone partitioned out of the aqueous phase and into the lipid phase, thus limiting availability of the inhibitor to plants. However, the amount of inhibitor reaching the cotyledons of tocopherolprotected mustard seedlngs was still in excess of the amount necessary to cause white foliage, but it failed to produce the effect. Tocopherol treatment did not prevent the 4-chloro-5-(dimethylamino)-2-(a, a, a-trifluoro-m-tolyl) -3 (2H) -pyridazinone-induced buildup of fatty acids in mustard cotyledons but did partially circumvent the effect in barley leaves. The amount of linolenic acid relative to linoleic acid was reduced in barley leaves and chloroplasts by 4-chloro-5-(dimethylamino)-2-(a,a,a-trifluoro-m-tolyl)-3(2H)-pyridazinone action and this effect was circumvented by tocopherol.The most potent of the several known inhibitors of chloroplast pigment formation (10) is 4-chloro-5-(dimethylamino)-2-(a,a,a-trifluoro-m-tolyl)-3(2H)-pyridazinone. Efforts to determine a physiological basis for this inhibition led us to evaluate certain chloroplast constituents for the circumvention of Sandoz 6706' actions. We observed that the protective metabolites were identical to some of the lipoidal materials found effective 'Abbreviations: GLC: gas-liquid chromatography; Sandoz 6706:by Stowe (17) Seeds were germinated in 9-cm Petri dishes on moist filter paper (three layers) for a period of 4 or 5 days. For evaluation of protective effects of fat-soluble (lipid) materials the filter paper was impregnated with 1, 10, or 100 ,tmoles of the compound dissolved in 1 or 2 ml of either acetone or petroleum ether (b.p. 30-60 C). After evaporation of the solvent, 10 ml of distilled water containing the inhibitor at indicated concentrations was added, and seeds were planted directly on the moist paper.Experiments to measure chlorophyll formation were conducted in a controlled environment room on a 15-hr photoperiod. The light source was a combination of fluorescent and incandescent lamps with an intensity of 1.5 x 10' ergs/cm'-sec. Day temperature was 27 C, and night temperature was 21 C, except as indicated otherwise. Approximately 70 mustard seeds were planted to insure 50 established seedlings p...