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, darkincubated 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 bisphosphates. 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 bicarbonate 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 MATERIALS AND METHODSThe 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 ...

Induction of Photosynthetic Carbon Fixation in Anoxia Relies on Hydrogenase Activity and Proton-Gradient Regulation-Like1-Mediated Cyclic Electron Flow in Chlamydomonas reinhardtii

“…However, some electrons originated from PSII might also be rerouted toward O 2 reduction (PSI-Mehler reaction, mitochondrial respiration, etc.). To test these possibilities, we added, prior to illumination, 2-hydroxyacetaldehyde (glycolaldehyde), an effective inhibitor of phosphoribulokinase (Sicher, 1984), or carbonyl cyanide m-chlorophenyl hydrazone (CCCP), an uncoupler of membrane potential preventing ATP synthesis in mitochondria and chloroplasts. In the presence of these inhibitors, both J H2 Table I.…”

Evidence for a Light Dependent Increase of Phosphoglucomutase Activity in Isolated, Intact Spinach Chloroplasts

“…Plants were raised from seed in controlled environment chambers providing 700 ,umol m-2 s-', PPFD, with a 9 h photoperiod, a 23°C day/18°C night cycle and 65% relative humidity. Plants were raised in large plastic trays containing vermiculite and were watered daily with complete mineral nutrient solution (22 Enzyme measurements were usually performed at 27°C by injecting 20 to 50 uL of the chloroplast suspension directly into mL reaction mixture containing 50 mM Hepes-NaOH (pH 7.6), 1 mM MgCl2, 0.2 mm NAD+, and unit of Glc6-P dehydrogenase from Leuconostoc mesenteroides. Assays were initiated with 2 mm Glcl-P and increased absorbance was measured at 340 nm.…”

The Mechanistic Basis of Internal Conductance: A Theoretical Analysis of Mesophyll Cell Photosynthesis and CO2 Diffusion

“…However, when the external CO 2 concentration was increased, the concentration difference was lower than expected, suggesting a possible loss of carbon from the stroma or a decrease of stroma pH (Werdan and Heldt, 1972;Sicher, 1984). In addition, Heber and Purczeld (1978) showed that even though the permeability of the chloroplast membrane to HCO 3 2 is very low, some The traditional resistance-based approach is compared with results determined from the reaction-diffusion model.…”