Mass-spectrometric determination of O2 and CO 2 gas exchange in illuminated higher-plant cells : Evidence for light-inhibition of substrate decarboxylations.

In order to estimate photosynthetic and respiratory rates in illuminated photoautotrophic cells of carnation (Dianthus caryophyllus L.), simultaneous measurements of CO2 and O2 gas exchange were performed using (18)O2, (13)CO2 and a mass-spectrometry technique. This method allowed the determination, and thus the comparison, of unidirectional fluxes of O2 and CO2. In optimum photosynthetic conditions (i.e. in the presence of high light and a saturating level of CO2), the rate of CO2 influx represented 75±5% of the rate of gross O2 evolution. After a dark-to-light transition, the rate of CO2 efflux was inhibited by 50% whereas the O2-uptake rate was little affected. The effect of a recycling of respiratory CO2 through photosynthesis on the exchange of CO2 gas was investigated using a mathematical model. The confliction of the experimental data with the simulated gas-exchange rates strongly supported the view that CO2 recycling was a minor event in these cells and could not be responsible for the observed inhibition of CO2 efflux. On the basis of this assumption it was concluded that illumination of carnation cells resulted in a decrease of substrate decarboxylations, and that CO2 efflux and O2 uptake were not as tightly coupled in the light as in the dark. Furthermore, it could be calculated from the rate of gross photosynthesis that the chloroplastic electron-transport chain produced enough ATP in the light to account for the measured CO2-uptake rate without involving cyclic transfer of electrons around PS I or mitochondrial supplementation.

Mass spectrometric determination of O2 gas exchange during a dark-to-light transition in higher-plant cells : Evidence for two individual O2-uptake components.

The exchange of O2 and CO2 by photoautotrophic cells of Euphorbia characias L. was measured using a mass-spectrometry technique. During a dark-tolight transition the O2 uptake rate was little affected whereas CO2 efflux was decreased by 40%. In order to differentiate eventual superimposed O2-uptake processes, the kinetics of O2 exchange resulting from brief illuminations were measured with a highly sensitive device. When the cells were exposed to a saturating light for short periods, the rate of O2 uptake passed through a series of transients: there was first a stimulation occurring 2-3 s after the appearance of O2 from water-splitting, followed 30 s later by an inhibition. These two transients were reduced 80% by 3-(3',4'-dichlorophenyl)1, 1-dimethylurea (DCMU), indicating that they relied on the linear transport of electrons in the chloroplasts. The first transient (stimulation of an O2 uptake) was little affected by mitochondrial inhibitors such as antimycin A and oligomycin or the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) but was increased in presence of KCN. When spaced flashes (2 us duration; 100-ms intervals) were used instead of continuous light, this transient was almost suppressed indicating that it was dependent on the saturation of some component of the chloroplastic chain. The second transient (inhibition of O2 uptake) was present when spaced flashes were used instead of continuous light. It was markedly decreased by addition of CCCP and mitochondrial inhibitors (antimycin A, oligomycin, KCN) which strongly indicates that it relied on mitochondrial respiration. It is concluded from these experiments that illumination of the cells resulted in an inhibition of mitochondrial respiration, but the resulting inhibition of O2 uptake was hidden by the appearance of an O2-uptake process of extramitochondrial origin, presumably located in the chloroplast.

Effects of Glucose Feeding on Respiration and Photosynthesis in Photoautotrophic Dianthus caryophyllus Cells: Mass Spectrometric Determination of Gas Exchange.

When glucose (20 millimolar) was added to photoautotrophic cell suspension cultures of Dianthus caryophyllus, there was during the first 10 hours an accumulation of carbohydrates and phosphorylated compounds. These biochemical changes were accompanied by a progressive decrease of net photosynthesis and a twofold increase of the dark respiratory rate. The rise of respiration was associated with a rise of fumarase and cytochrome c oxidase activities, two mitochondrial markers. Gas exchange of illuminated cells were performed with a mass spectrometry technique and clearly established that during the first hours of glucose feeding, the decrease of net photosynthesis was essentially due to an increase of respiration in light, whereas the photosynthetic processes (gross O(2) evolution and gross CO(2) fixation) were almost not affected. However, after 24 hours of experiment, O(2) evolution and CO(2) fixation started to decline in turn. While ribulose-1,5-bisphosphate carboxylase activity was little affected during the first 48 hours of the experiment, the maximal light-induced phosphoribulokinase activity dramatically decreased with time and represented after 48 hours only 30% of its initial activity. It is postulated that the decrease in phosphoribulokinase activity was at least partially responsible for the decrease of CO(2) fixation and the metabolic events involved in this regulation are discussed.