Oxygen Concentration in Isolated Chloroplasts during Photosynthesis

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...

“…2). Experi- [30].) This 02 affimity is similar to that previously determined for algae (22,26), but considerably lower than that reported from experiments with broken chloroplasts (2,12).…”

supporting

confidence: 53%

Photosynthetic O₂ Exchange Kinetics in Isolated Soybean Cells

Behrens¹,

Marsho²,

Radmer³

1982

“…The sampling system used in these experiments does not necessitate equilibration across a gas-liquid interface, but incomplete equilibration of the 02 isotopes between the inside and outside of the chloroplasts (or cells) will result in an underestimation of the rate of 02 reduction. However, the relatively simpler isolated chloroplast system and the reported (27) …”

Section: Discussionmentioning

confidence: 99%

Photosynthetic Oxygen Reduction in Isolated Intact Chloroplasts and Cells in Spinach

Marsho¹,

Behrens²,

Radmer³

1979

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

“…We found that lamellae 02 reduction was saturated fully with respect to 02 levels as low as 155 /IM and our measurements yielded Km values of 13.5 to 22.2 ,uM (24). 02 concentrations in the illuminated intact chloroplast may rise as high as I mm (29), such that in vivo it is likely that the sites where 02 may bind to be reduced are strongly competed for by other reducible moieties, e.g. NADP and nitrite.…”

Section: Discussionmentioning

confidence: 99%

Hydrogen Peroxide Synthesis in Isolated Spinach Chloroplast Lamellae

Robinson¹,

Gibbs²

1982

Light-dependent 02 reduction concomitant with 02 evolution, ATP formation, and NADP reduction were determined in isolated spinach (Spinacia oleracea L. var. America) chloroplast lamellae fortified with NADP and ferredoxin. These reactions were investigated in the presence or absence of catalase, providing a tool to estimate the reduction of 02 to H202 (Mehler reaction) concomitant with NADP reduction. In the presence of 250 micromolar 02, 02 photoreduction, simultaneous with NADP photoreduction, was dependent upon light intensity, ferredoxin, Mn2", NADP, and the extent of coupling of phosphorylation to electron flow.In the presence of an uncoupUng concentration of NH4', saturating Ught intensity (>500 watts/square meter), saturating ferredoxin (10 micromolarity) rate-limiting to saturating NADP (0.2-0.9 milimolarity), and Mn2+ (50-1000 micromolarity), the maxium rates of 02 reduction were [13][14][15][16][17][18][19][20][21][22][23][24][25] H202 (18, 21, 30) and this reductive reaction is associated with PSI activity coupled to noncyclic electron transport (10,15,18,21,23,30). In preparations of NADP-deficient chloroplast lamellae, it has been established that 02 photoreduction results in the forma- (Mehler reaction) (4,(11)(12)(13)19). Electron flow from H20 through the photosystems to 02 was shown to be associated with ATP synthesis and was designated as pseudocyclic photophosphorylation (1, 3, 15). However, recent evidence has indicated that the primary function of 02 reduction is to prevent over-reduction and proper poising of redox states for components of the photosynthetic electron transport chains (3,16,28,30, 32). Additionally, current evidence strongly suggests that 02 serves to regulate interaction between the cyclic and noncyclic electron transport systems (3,16,28, 32).One of the questions which has remained is whether or not 02 photoreduction can be observed with chloroplast preparations in the presence of rate-limiting as well as saturating levels of NADP. The experiments of Elstner et al. (12) indicated that in isolated spinach plastid lamellae, fortified with NADP and Fd, there was not a measurable light-dependent formation of 02-or H202 until 85 to 90%o of the total NADP supplied had been reduced. This observation implied that, when NADP levels were saturating, there was no competition between NADP and 02 for reducing sites on the enzyme Fd-NADP reductase. In contrast, Allen (1) observed a measurable 02 photoreduction to H202 in plastid lamellae supplied with Fd and NADP, and these data were interpreted to mean that there existed a constant competition between NADP and 02 for reducing equivalents.In this report, we present evidence that there is a significant reduction of 02 to H202 concomitant with NADP photoreduction in lamellae fortified with NADP and Fd. We show that the rate of02 reduction concomitant with NADP reduction was dependent upon light intensity, Fd, Mn2 , and NADP and was affected by an uncoupling agent. We also demonstrate and discuss the influence of the Mehler re...