Photosynthetic Oxygen Reduction in Isolated Intact Chloroplasts and Cells in Spinach

Abstract

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

Evidence for Cyclic Photophosphorylation during 14CO2 Fixation in Intact Chloroplasts

“…Marsho et al (18) found that, although there was a significant rate of 02 uptake in intact chloroplasts during the lag phase of photosynthesis, this diminished to a relatively insignificant level during steady-state photosynthesis. These low rates of 02 uptake were relatively insensitive to increases in light intensity which greatly stimulated photosynthesis (14).…”

“…Thus, the relative contribution of cyclic versus pseudocyclic flow to the ATP/NADPH balance during photosynthesis remains unresolved. Furthermore, the reasons for the discrepancies between high (8, 9) and low (14,18) rates of 02 uptake measured during photosynthesis in chloroplasts and cells remain to be resolved.…”

Responses of Superoxide Dismutase and Glutathione Reductase Activities in Cotton Leaf Tissue Exposed to an Atmosphere Enriched in Oxygen

“…Further uptake of molecular O2 and its reduction to H202 in isolated chloroplasts, the Mehler reaction, has been considered to be a component of pseudocyclic electron transport in algae and higher plants. This process, however, occurs to a significant extent in higher plants only if conditions are not optimal for normal CO2 fixation (19). Metabolism of 02 is initiated by a univalent reduction to superoxide free radical; dismutation of superoxide anions catalyzed by superoxide dismutase (EC 1.…”

Tansley Review No. 112

“…Rates of electron transport to O # might increase transiently during light flecks, when rapid adjustment of regulation between the processes of electron transport and CO # assimilation is required (Pearcy, 1990). This situation is analogous to the induction period of photosynthesis (Marsho et al, 1979 ;Foyer et al, 1992). Although it has frequently been considered that high light intensities promote higher rates of O # reduction (Canvin et al, 1980 ;Badger, 1985 ;Grace & Logan, 1996), it has also been suggested that O # reduction accounts for a greater proportion of electron flow at low light intensities (Heber et al, 1978).…”

“…Likely values for flux through the Mehlerperoxidase cycle in planta are not known and estimated rates vary enormously (Marsho et al, 1979 ;Steiger & Beck, 1981 ;Badger, 1985 ;Robinson, 1988). Mass spectrometric measurements are complicated by concomitant O # uptake in photorespiration and mitochondrial respiration (sections V and VI).…”