Studies on Effects of Certain Quinones

Abstract

The effect of quinone herbicides and fungicides on photo-synthetic reactions in isolated spinach (Spinacia oleracea) chloroplasts was investigated. 2, 3-Dichloro-1, 4-naphthoqui-none (dichlone), 2-amino-3-chloro-1,4-naphthoquinone (06K-quinone), and 2,3,5,6-tetrachloro-1 ,4-benzoquinone (chlor-anil) inhibited ferricyanide reduction as well as ATP formation. Benzoquinone had little or no effect on these reactions. The two reactions showed a differential sensitivity to these in-hibitors. Dichlone was a strong inhibitor of both photosystems I and II; photosystem I was more sensitive to 06K-quinone than was photosystem II, whereas the reverse was true of chloranil. Chloranil and 06K-quinone inhibited ferricyanide reduction and the coupled photophosphorylation to the same extent, whereas dichlone affected photophosphorylation to a greater extent than the ferricyanide reduction. CO2 fixation was inhibited by aUl the quinones to varying degrees. In chloroplasts treated with 06K-quinone or benzo-quinone, CO2 fixation was inhibited to a greater extent than the photoreduction of ferricyanide or ATP formation, indicating the possibility that the two quinones may also inhibit certain reactions in the carbon reduction cycle. The effect of dichlone and chloranil, but not of 06K-quinone, was overcome by the addition of reduced glutathione. The quinones caused an increase in the proportion of "4C incorporated into 3-phosphoglyceric acid and a reduction in the amount of glycolic acid. A number of quinonoid compounds have been found to have fungicidal, algicidal, and herbicidal effects. The fungicidal activity of substituted benzoquinones and naphthoquinones was studied and has been found to be related to the inhibition of enzymes and coenzymes with free sulfhydryl and amino groups (11, 14). Very little information is available on the mode of action of quinone algicides or herbicides in algae and higher plants. Zweig et al. (16, 17) reported that certain quinones, herbicides, and algicides inhibited oxygen evolution and CO2 fixation in Chlorella, suggesting that these chemicals interfere with photosynthesis. In order to elucidate which site(s) in the ' photosynthetic process is affected by the quinones, the effect of these inhibitors on photosynthetic reactions such as electron transport, photophosphorylation, and CO, fixation was investigated in isolated chloroplasts. The quinones tested in this study included 2,3-dichloro-1,4-naphthoquinone, 2-amino-3-chloro-1, 4-naphthoquinone, 2, 3, 5, 6-tetrachloro-1 , 4-benzo-quinone, and 1,4-benzoquinone. MATERLALS AND METHODS For electron transport and photophosphorylation studies, the chloroplasts were prepared from spinach (Spinacia olera-cea) leaves as described by Avron (2). The chloroplasts were washed once with the sucrose-tris-NaCl homogenizing medium without ascorbate. Chlorophyll was determined by the method of Arnon (1). Measurement of Fefficyanide Reduction and Photophospho-rylation. The effect of quinones on the photoreduction of fer-ricyanide was measured under both nonphosphorylating (ba-sal) and phosphorylating conditions. The procedure followed for the measurement of photophosphorylation was as described by Avron (2). The standard reaction mixture contained, in ,umoles: tris, pH 7.8, 45; NaCl, 60; MgCL,, 12; sodium-potassium phosphate, pH 7.8, 12; ADP, 12; adequate 'P (about 106 cpm); PMS3, 0.09 (cyclic photophosphorylation); or potassium ferricyanide, 0.5 (noncyclic); chloroplast suspension containing between 25 and 50 ,ug of chlorophyll, quinone solution, and water to a total volume of 3.0 ml. In the measurements of basal electron transport, phosphorylating reagents (ADP, Mg2+, and P,) were omitted from the reaction mixture. The tubes containing the reaction mixtures were placed in a water bath maintained at 15 C, and the reaction was started by turning the light on. The light intensity at the level of the tube was approximately 16,000 ft-c. After 2 min of illumination, the light was turned off, and each chloroplast suspension was inactivated quickly by the addition of 0.3 ml of 30% (w/v) trichloro-acetic acid. The samples were centrifuged, and aliquots of supernatant were analyzed for 'P (ATP), as described by Avron (2). Ferricyanide reduction was determined by measuring the absorption at 420 nm in centrifuged aliquots of the reaction mixture. P/ 2e ratio was calculated from the values of ferri-cyanide reduction and ATP formation. CO2 Fixation. For CO2 fixation studies, chloroplasts were isolated from 4-to 6-week-old spinach grown in the field. The leaves were picked immediately prior to their use in experi-3 Abbreviations: chloranil: 2, 3 , 5, 6-tetrachloro-1, 4-benzoqui-none; dichlone: 2, 3-dichloro-1,4-naphthoquinone; 06K-quinone: 2-amino-3-chloro-1 ,4-naphthoquinone; PMS: phenazine methosul-fate.