Photorespiratory Properties of Mesophyll Protoplasts of Nicotiana plumbaginifolia

The photorespiratory activity of mesophyll protoplasts of Nicotlana plumbaglnhfolla has been clearly demonstrated by the presence of a Warburg-effect, the occurrence of an important C02-sensitive 02 uptake and the effect of some photorespiratory inhibitors on photosynthetic activity. At a nonsaturating dissolved inorganic carbon (DIC) concentration (0.1 millimolar), we observed that the rate of CO2 fixation was 60% lower at 50% 02 compared to that measured at 2% 02. Using 1802 and mass spectrometry, we measured 02 exchange as a function of light intensity and of DIC concentration. Oxygen uptake measured at the CO2 compensation point (47.4 micromoles 02 per hour per milligram chlorophyll) was three-fold higher than that measured at a saturating CO2 concentration. Cyanide or iodoacetamide, inhibitors of the Calvin cycle, were found to reduce the 02 uptake to the same extent as CO2 saturation. We conclude from these results that the major part of the C02-sensitive 02 uptake is due to photorespiration. Further, we investigated the effect on net photosynthesis of some inhibitors of the glycolate pathway. At CO2 saturation (10 millimolar DIC), 5 millimolar aminoacetonitrile (AAN), and I millimolar aminooxyacetate (AOA) did not cause any significant decrease in net photosynthesis. However, when these two inhibitors were added under a period of active photorespiration (10 minutes at the CO2 compensation point at 20% 02), we observed a decrease in the rate of net photosynthesis at 10 millimolar DIC measured afterward (respectively, 18 and 29%). This inhibition did not appear at 2% 02, but was stronger at 50% 02 (40% for AAN and 47% for AOA). With 0.05 millimolar butyl 2hydroxy-3-butynoate (BHB) or 0.5 millimolar L-methionine-DL-sulfoximine (L-MSO), rates of net photosynthesis at 10 millimolar DIC were decreased by 10 to 15%. Additional decreases were observed after a period at the CO2 compensation point at 20% 02 (30% for BHB and 20% for L-MSO). From the sites of action of the four inhibitors tested, we suggest the inhibition of photosynthesis occurring after a period of active photorespiration to be due to the toxic accumulation of nonmetabolized phosphoglycolate. tion between 02 and CO2 at the active site of Rubisco,' the enzyme catalyzing the initial reactions ofboth photosynthetic and photorespiratory metabolisms. In other experiments, the activity of the photosynthetic carbon oxidation cycle was deduced from the effect on photosynthesis of some photorespiratory inhibitors. AAN, an inhibitor of the conversion of glycine to serine, has been shown to cause a decrease in the photosynthetic activity in protoplasts of Triticum aestivum (29), only when added in conditions of active photorespiration. Such an inhibition is usually attributed to the accumulation ofnonmetabolized photorespiratory intermediates. According to Servaites and Ogren (27), the inhibition of photosynthesis would result from a deficiency in the recycling of carbon in the Calvin cycle, but Creach and Stewart (5) did not observe a decrease in the RubP level in leaves of Glycine max in the presence ofAAN and they concluded to a toxicity of accumulated intermediates. In the present work, we have further studied the photorespiratory properties of mesophyll protoplasts of Nicotiana plumbaginifolia using a direct method of02 exchange measurement based on MS. We have determined the effect ofCO2 concentration and of inhibitors of the Calvin cycle on 02 exchange. We conclude from our results that most of the C02-sensitive 02 uptake in the light is due to photorespiration. We have also investigated the effect on net photosynthesis of some inhibitors acting at different steps of the glycolate pathway (AAN, AOA, BHB, and L-MSO) under various photorespiratory conditions. MATERIAL AND METHODS