The time course of light-induced 02 exchange by isolated intact chloroplasts and cells from spinach was determined under various conditions using isotopicaUy labeled 02 and a mass spectrometer. In dark-adapted chloroplasts and ceUs supplemented with saturating amounts of bicarbonate, 02 evolution began immediately upon ilumination. However, this initial rate of 02 evolution was counterbalanced by a simultaneous increase in the rate of 02 uptake, so that little net 02 was evolved or consumed during the first 1 I minute of illumination. After this induction (lag) phase, the rate of 02 evolution increased 3 to 4-fold while the rate of 02 uptake diminished to a very low level. Inhibition of the Calvin cycle, e.g. with DLglyceraldehyde or iodoacetamide, had negligible effects on the initial rate of 02 evolution or 02 uptake; both rates were sutained for several minutes, and about balanced so that no net 02 was produced. Uncouplers had an effect similar to that observed with Calvin cycle inhibitors, except that rates of 02 evolution and photoreduction were stimulated 40 to 50%.These results suggest that higher plant phostosynthetic preparations which retain the ability to reduce CO2 also have a significant capacity to photoreduce 02. With (5,6,12,14,15,17). Here, we determined the magnitude and kinetics of 02 evolution and reduction in isolated intact chloroplasts and whole cells obtained from spinach using a mass spectrometer with a fast mass stepper system. Significant rates of 02 reduction are shown to occur principally when CO2 fixation reactions are suboptimal. MATERIALS AND METHODSIntact chloroplasts were isolated from greenhouse-grown spinach as described previously (26). Preparations contained more than 70%/o intact chloroplasts as determined by the ferricyanide reduction method (13) and fixed CO2 at rates in excess of 100 ,umol/mg Chl * h in saturating light. Chloroplasts were routinely assayed in a 0.33 M sorbitol, 50 mm Hepes-KOH (pH 8.0) medium containing 10 mm NaHCO3, 5 mm Na4P207, 2 mm EDTA, 0.25 mM K2HPO4, and catalase (195 units/ml). Other additions are indicated in the figure legends.Intact cells were prepared from freshly harvested spinach leaves sliced into small (0.5 x 2.0 cm) strips. Approximately 2 g of leaf strips were vacuum-infiltrated in 20 ml of media containing 0.8 M sorbitol, 20 mm Mes (pH 5.8) buffer, 12.5 mm K2SO4, and 0.75% Macerase (obtained from Calbiochem). Leaf strips were digested in 75 ml of the infiltration medium (maintained at 15 C) in an apparatus similar to that described by Servaites and Ogren (23

Light-dependent 02 exchange was measured in intact, isolated soybean (Glycine max. var. Williams) cells using isotopicaHly labeled 02 and a mass spectrometer. The dependence of 02 exchange on 02 and C02 was investigated at high light in coupled and uncoupled cells. With coupled cells at high 02, 02 evolution followed similar kinetics at high and low C02. Steady-state rates of 02 uptake were insignificant at high C02, but progressively increased with decreasing C02. At low C02, steady-state rates of 02 uptake were 50% to 70% of the maximum C02-supported rates of 02 evolution. These high rates of 02 uptake exceeded the maximum rate of 02 reduction determined in uncoupled cells, suggesting the occurrence of another light-induced 02-uptake process (Le. photorespiration).Rates of 02 exchange in uncoupled cells were half-saturated at 7% to 8% 02-Initial rates (during induction) of 02 exchange in uninhibited cells were also half-saturated at 7% to 8% 02. In contrast, steady-state rates of 02 evolution and 02 uptake (at low C02) were half-saturated at 18% to 20% 02. 02 uptake was significantly suppressed in the presence of nitrate, suggesting that nitrate and/or nitrite can compete with 02 for photoreductant.These results suggest that two mechanisms (02 reduction and photorespiration) are responsible for the light-dependent 02 uptake observed in uninhibited cells under C02-limiting conditions. The relative contribution of each process to the rate of 02 uptake appears to be dependent on the 02 level. At high 02 concentrations (I-40%), photorespiration is the major 02-consuming process. At lower (ambient) 02 concentrations (c20%), 02 reduction accounts for a significant portion of the total light-dependent 02 uptake.It is generally recognized that one of the important factors limiting net photosynthesis in higher C3 plants under ambient conditions is the prevailing CO2 and 02 tensions (13,31,32). Decreasing CO2 or increasing 02 concentrations (from ambient) leads to significant decreases in the net rate of photosynthesis. Conversely, the net rate of photosynthesis can be increased by increasing the CO2 or decreasing the 02 concentrations. These observations have been interpreted as a reflection of the direct and/or indirect competition between CO2 and 02 for photosynthetic-reducing equivalents. Three principal 02-consuming represent a relatively minor component of the total 02 uptake (see, however, 9, 10) and most attention has focused on the lightdependent processes of 02 reduction and photorespiration. The extent to which each process occurs in vivo under different photosynthetic conditions is unresolved.Endogenous 02 reduction has been shown to occur in a variety of photosynthetic systems (2,8,11,17,21,29). This light-dependent process is eliminated by DCMU, is absent in PSI-deficient algal mutants (24), and is not directly dependent on reactions of the carbon reduction cycle. Inhibition of 02 reduction by CO2 presumably reflects the competition between 02 and NADP+ for photosynthetic reducing equivalents. 02 r...

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