Characteristics of Light-Dependent Inorganic Carbon Uptake by Isolated Spinach Chloroplasts

Abstract

The light-dependent accumulation of radioactively labeled inorganic carbon in isolated spinach (Spinacia orwcea L.) chloroplasts was determined by silicone oil filtering centrifuption. Intact chloroplasts, dark-incubated 60 seconds at pH 7.6 and 23°C with 0.5 millimolar sodium bicarbonate, contained 0.5 to 1.0 millimolar internal inorganic carbon. The stromal pool of inorganic carbon increased 5-to 7-fold after 2 to 3 minutes of light. The saturated internal bicarbonate concentration of illuminated spinach chloroplasts was 10-to 20-fold greater than that of the external medium. This ratio decreased at lower temperatures and with increasing external bicarbonate. Over one-half the inorganic carbon found in intact spinach chloroplasts after 2 minutes of light was retained during a subsequent 3-minute dark incubation at 5'C. Calculations of light-induced stromal alkalization based on the uptake of radioactively labeled bicarbonate were OA to 0.5 pH units less than measurements performed with '4qCdimethyloxazolidine-dione. About one-third of the binding sites on the enzyme ribulose 1,5-bisphosphate carboxyase were radiolabeled when the enzyme was activated in situt and 4C02 bound to the activator site was trapped in the presence of carboxypentitol bis-phosphates. Deleting orthophosphate from the incubation medium eliminated inorganic carbon accumulation in the stroma. Thus, bicarbonate ion distribution across the chloroplast envelope was not strictly pH dependent as predicted by the Henderson-Hasselbach formula. This finding is potentially explained by the presence of bound CO2 in the chloroplast. that illumination should produce a 10-fold increase of bicarbon-ate ion within this space. The following report details efforts in this laboratory to verify this observation. In addition to being the substrate for photosynthetic carbon assimilation, CO, is an essential factor in the light-activation of the enzyme RuBP' carboxylase (14, 18). Results obtained with isolated enzyme preparations demonstrated that an activator CO2 molecule, which is not the substrate for CO2 fixation, forms an e-aminocarbamate with a lysyl residue located near the active site region (18). Because the carboxylase constitutes over one-half the soluble protein in the stroma, the concentration of CO2 binding sites in the chloroplast exceeds 3 mm (14). The fractional occupancy of these binding sites is unknown (4), although recent evidence suggests that RuBP carboxylase in vivo is predominantly activated at saturating irradiances for photosynthesis (19, 23). Direct evidence for high concentrations ofCO2 bound to protein has been obtained via '3C-NMR analysis of Euglena gracilis Z cells (29). Sequestering inorganic carbon as a protein-carbamate may prevent the diffusive loss of CO2 from the cell. It is obvious that protein-bound CO2 would effect the distribution ofinorganic carbon across the chloroplast envelope independent of the Hen-derson-Hasselbach relationship and would complicate stromal pH determinations using bicarbonate salts. MATERIALS AND METHODS The low ambient CO2 concentration in the atmosphere may be a critical factor limiting the photosynthetic rate of agronomic crop species (6). In spite of the importance of CO2 to the carbon assimilation process, there have been relatively few studies on the acquisition of inorganic carbon by isolated chloroplasts or by chloroplast envelope preparations (21, 22, 27). The transfer of inorganic carbon across the chloroplast membrane has been reviewed recently (8) and this process has the following important features: (a) permeability of the chloroplast envelope is high for CO2 relative to bicarbonate (27), (b) diffusion of CO2 across the chloroplast envelope is rapid and bicarbonate accumulation saturates in less than 30 s at 9C (21, 27), and (c) distribution of bicarbonate ion across the chloroplast envelope is dependent upon the pH gradient between the internal and external medium as predicted by the Henderson-Hasselbach equation (27). This latter observation is significant inasmuch as the internal and external bicarbonate ion concentrations can be used to calculate the pH of the chloroplast stroma (27). It is widely recognized that illumination of isolated, intact chloroplasts induces an al-kalization of the stroma ofabout 1.0 pH unit (12, 28), suggesting Chloroplast Isolation. Intact spinach chloroplasts were isolated from 4-to 5-week-old spinach plants (Spinacia oleracea var. Viroflay, Ferry Morse Seed Co., Mountain View, CA)2 as described previously (15). Plants were raised from seed in vermi-culite in controlled environment chambers (800 ,E m 2 s-', 9-h photoperiod, 22C day/18C night, 65 ± 5% RH) and were watered daily with complete nutrient solution. Chloroplast integrity exceeded 80% when determined by ferricyanide-dependent 02 evolution (9). Chl was assayed in 80% (v/v) aqueous acetone (1). Bicarbonate Uptake. Accumulation ofinorganic carbon in the chloroplast stroma was determined by silicone oil filtering cen-trifugation (27). A 0.2 ml suspension of chloroplasts (0.008 to 0.018 mg Chl) in solution C (15) which contained 330 mM sorbitol, 50 mM Hepes-NaOH (pH 7.60), 2 mm EDTA, 1 mM MgCl2, 1 mM MnCl2, and 0.5 mm K2HPO4 (or as indicated) was 'Abbreviations: RuBP, ribulose 1,5-bisphosphate; Ci, internal inorganic carbon; C., external inorganic carbon; CPBP, carboxypentitol bisphosphates; DMO, 5,5-dimethyloxazoladine-2,4-dione; NMR, nuclear magnetic resonance. 2 Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. 962