Photosynthetic Oxygen Exchange in Isolated Cells and Chloroplasts of C 3 Plants

Abstract

Photosynthetic 02-production and photorespiratory 02-uptake were measured, using stable isotope techniques, in isolated intact leaf cells of the C3 plant Xanthium strumariun L., and isolated intact chloroplasts of Spinacia okracea L (var. Yates 102). Considerable light dependent 02-uptake was observed in both systems, a proportion of which could be suppressed by CO2 (63% suppression in chloroplasts by 50 micromolar C02, 58% in cells by 100 micromolar CO2 and 250 micromolar O2). At low 02, 02-uptake was CO2 insensitive. At high CO2 up to 19% of total electron flow was to 02 in cells and up to 14% in chloroplasts. 02-uptake showed inhibition by KCN (61% in cells, 35% in chloroplasts by 0.2 miimolar KCN). 02-uptake half saturated at 75 to 85 micromolar 02 in cells and 50 to 65 micromolar 02 in chloroplasts, at low CO2. The results are discussed in terms of the RuP2-oxygenase reaction and direct photoreduction of 02 via a Mehler reaction. Light-dependent 02 uptake has been recognized as a feature of photosynthesis since the early observations of Hoch et al. (15) using isotopic 02. The components of this 02 uptake are believed to include RuP2-oxygenase' (20), direct photoreduction of 02 via a Mehler reaction (1, 12) or, alternatively, persistence of mitochondrial respiration during illumination (1 1). Varying views have been expressed as to the relative magnitudes of the above uptake processes under a range of conditions for photosynthesis. Investigation into this area would provide data pertinent to the role of 02 in two metabolic events. First, there has been considerable discussion concerning the relative roles of RuP2-oxygenase versus the Mehler reaction and its photochemically generated H202 as the primary event of photorespiration (29). Second, there has been controversy over the relative magnitudes of cyclic and pseudocycic (whole chain electron transport to O2-Mehler reaction) photophosphorylation as mechanisms to generate the additional ATP required for CO2 fixation and photores-piration (12). The present studies address the above problems by examining the effects of CO2 and O2 concentration on the O2 exchange (photosynthetic 02-evolution and uptake) of isolated cells and chloroplasts in order to define the parameters of 02 uptake in these systems. MATERIALS AND METHODS Spinach chloroplasts were prepared by the method of Lilley and Walker (18) from 5-week-old leaves of Spinacia oleracea L. ' Abbreviation: RuP2-oxygenase, ribulose-1,5-bisphosphate oxygenase (EC 4.1.1.39). (var. Yates 102) glasshouse grown in water culture. Leaf cells from Xanthium strumarium (glasshouse grown in soil) were isolated according to Sharkey and Raschke (26), and isolation was completed in less than 5 min after leaf detachment. 02 exchange was measured using a Varian MAT GD 150/4 magnetic sector mass-spectrometer, continuously monitoring 1802 (mass 36), 1602 (mass 32), and argon (mass 40) as an internal reference gas (this machine has four collectors allowing each mass to be collected separately and simultaneously). Cells and chloroplasts were placed in a glass cuvette (similar in design to that of an 02-electrode) in media depleted of 02 by bubbling with argon. The cuvette was stoppered, and a bubble of 1802 was allowed to dissolve into the aqueous medium until the desiredfinal total 02 concentration (1802 + 1602) had been reached. The bubble was then removed, and the experimental measurements of 1602 and 1802 changes were started. Gases were admitted to the analyzer by diffusion across a polythene membrane set in the base of the cuvette. Calibration of mass signals with regard to concentration of species in solution was made by bubbling liquid in the cuvette with known gas concentrations (i.e. 100%o argon and 21% 1602 in air-as the signal response of the mass spectrometer is linear, then a single point calibration can be made). Temperature was controlled through a water jacket maintained at 25°C. Calculations of 02-uptake and evolution were carried out as given by Radmer and Kok (24) and Figure 1, with corrections based on the change in the argon signal for consumption of gas by the mass spectrometer. Leaks into and out of the cuvette to the atmosphere were negligible compared to the changes in signal levels caused by the photosynthetic reactions and mass spectrometer consumption. An example of a recorder tracing of the signal changes of masses 32, 36, and 40 following switching the light on is given in Figure 1. Cells were assayed in 0.1 M Hepes adjusted to pH 7.0 with KOH, and chloroplasts were assayed in 0.33 M sorbitol, 1 mm MgCl2, 2 mi EDTA, 50 mm Hepes, and 0.5 mm KH2PO4, adjusted to pH 7.6 with KOH. Rates of 02 exchange were measured after a linear rate was obtained unless otherwise specified. 02 concentrations indicated and O2 exchange rates were measured simultaneously ; total 02 varied less than 20%o throughout an experiment. CO2 concentrations indicated in the figures are initial values calculated from the added NaHCO3 and the pH. Chl concentration ranged from 3 to 10 ,ug/ml in a 5 ml volume. Chl was estimated by the method of Arnon (3). Chloroplasts were 80 to 95% intact as measured by ferricyanide reduction (13). All chemicals were obtained from Sigma with the exception of 1802 (99% enrichment) which was obtained from Norsk-Hydro (Oslo). RESULTS 02 uptake in cells and chloroplasts is considerably inhibited by increasing CO2 concentrations. Figure 2 illustrates this effect of CO2 concentration on 02-uptake and 02-evolution in spinach 927