The phytotoxic effect of Cu via the photosynthetic electron transport system was studied with isolated spinach chloroplasts. Cu(II) ions Induce a Ught-iven peroxidation of membrane lpids leading to ethylene formadon, the latter dominating over a concurrent ethane production. Seemingly, the hydroxyl radical originating from superoxide anion is the starting reactive 02 species. Cu ions inhibit photosynthetic electron transport and apparently catabze the formation of hydroxyl radical and Fenton-type reactions that result in destruction of unsaturated membrane fatty acids. 2Abbreviations: MV (methylviologen), 1,1'-dimethyl-4,4'-dipyridylium dichloride; DAD (diaminodurene), 2,3,5,6-tetramethyl-p-phenylenediamine; DCIP, 2,6-dichlorophenolindophenol; DCMU, 3-(3,4-dichlorophenyl)l,I-dimethylurea; SOD, superoxide dismutase DABCO, 1,4-diazobicyclo-2,2,2-octane.
MATERIALS AND METHODSChloroplasts from fresh spinach leaves (Spinacia oleracea, strain Atlanta) grown in the open during fall were prepared according to reference 4 and suspended in 50 mm phosphate buffer (pH 7.5), including 5 mm MgCl2. To obtain chloroplasts with high endogenous O2 uptake (as in Table V), the harvested leaves were stored at room temperature for 8 h prior to chloroplast preparation.Chloroplast incubation for the determination of either the lightinduced formation of C2H6, and C2H4 or O2 was carried out in a Warburg apparatus (Braun, Melsungen) using 150 w/m2 light, provided by simple tungsten light bulbs. The reaction mixture to measure O2 generation included, in a 2-ml reaction volume: 50 mim phosphate buffer (pH 7.5), 5 miM MgCl2, 5 mm NH4Cl, 0.5 mM hydroxylamine, and chloroplast material equivalent to 100 ,ug Chl. The time of incubation was 10 min. Then, the nitrite formation from the added hydroxylamine due to the presence of superoxide was measured as described (9).The medium for production of C2H6 and C2H4 contained 10 mm phosphate buffer (pH 7.5), 5 mM MgCl2, 5 mm NH4Cl, and chloroplasts equivalent to 200 jig Chl in a 2-ml final reaction volume. Incubation was over a period of 1 h in 10-ml vessels sealed with gas-tight rubber caps. The separation of both gases was performed with a 2-m column of activated alumina using a temperature program from 60 to 140 C and a gas chromatograph (model F22, Perkin-Elmer) equipped with an automatic headspace sampler (20).Photosynthetic NADP+ and DCIP reduction was performed and measured continuously in 50 mm phosphate buffer (pH 7.5), including 5 mM MgCl2 and 5 mm NH4Cl, using a split-beam spectrophotometer (Hitachi, model 124) equipped with a 40-w tungsten lamp for actinic cross-illumination. In addition, the H20 --NADP+ reaction mixture contained 0.4 mi NADP+ and 2 uM Bumilleriopsis ferredoxin; with DAD as electron donor, this assay included additionally 1 mm sodium ascorbate, 100 tLM DAD, and 100 /M DCMU. The DCIP concentration was 50 tiM when using the H20 --DCIP system. 02 uptake of isolated chloroplasts was measured with a Clarktype 02 electrode with red light (140 w/m2) using an RG 610 Schott c...
