Photosynthetic Oxygen Reduction in Isolated Intact Chloroplasts and Cells in Spinach

Abstract

The time course of light-induced 02 exchange by isolated intact chlo-roplasts 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 bicarbon-ate, 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 DL-glyceraldehyde 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 near-saturating Ught and sufficient C02, 02 reduction appears to take place primarily via a direct interaction between 02 and reduced electron transport carriers, and occurs principally when CO2-fixation reactions are suboptimal, e.g. during induction or in the presence of Calvin cycle inhibitors. The inherent maximum endogenous rate of 02 reduction is approximately 25 to 50% of the maximum rate of noncycic electron transport coupled to CO2 fixation. Mthough the photoreduction Of 02 is coupled to ion transport and/or phosphorylation, this process does not appear to supply significant amounts of ATP directly during steady-state CO2 fixation in strong light. Efficient photosynthetic CO2 fixation is dependent upon the light-driven production of NADPH and ATP in appropriate stoichiometric amounts. One means for assuring that correct stoi-chiometries are maintained would be to vary the rate of photosyn-thetic electron flow through pathways differing in their ATP: NADPH production ratios (10). The possible occurrence of coupled electron flow pathways other than the noncycic transport from water to NADPH, e.g. pseudocycic or cyclic electron transport , has been recognized for some time (2, 16). The extent to ' This research was supported in part by grants from the National Sciences Foundation (GB-38237), USDA/SEA Competitive Grants Office (7801019) (TVM), and USDA/SEA Competitive Grants Office (5901-0410-8-0179-0) Department of Energy (EY-76-C-02-3326) (RR). which these pathways operate in vivo, however, is still unresolved. Studies with washed broken chloroplast preparations have shown that rates of electron transport to 02 are generally quite low unless certain exogenous electron acceptors are added. In contrast, algae or intact isolated chloroplasts display a potentially significant endogenous capacity for pseudocyclic (and cyclic) transport (see, e.g. 1, 8, 19, 20, 24). Under certain conditions, algae have been shown to carry out pseudocyclic electron transport (02 reduction) at rates approaching saturated rates of noncycic electron transport (24). A number of direct and indirect measurements with isolated intact chloroplasts have suggested that higher plant photosynthetic tissues also possess a substantial endogenous capacity for 02 reduction (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 METHODS Intact 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). Released cells were pelleted (100g), washed twice with 50 mm Hepes-KOH (pH 7.8) buffer containing 0.8 M sorbitol and 1 mM MgCl2, and subsequently resuspended in a small volume of the washing medium containing 5 mM DTT. Preparations contained 2 85% intact (plasmolysed) cells as judged microscopically after staining with 2.5% Evans Blue. Cells were normally assayed in a medium consisting of 0.7 M sorbitol, 50 mM Hepes-KOH (pH 8.0) buffer, 10 mm NaHCO3, and 1 mm MgCl2. Saturated rates of net 02 evolution were routinely 50 to 70 ,umol 02/mg ChI * h. 02 exchange was measured polarographically or with a mass spectrometer using isotopically labeled 02 (99 atom% 1802, obtained from Bio-Rad Laboratories). The instrumentation and 656 www.plantphysiol.org on May 24, 2017-Published by www.plantphysiol.org Downloaded from