Fixation of 18O2 during Photorespiration

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Abstract
Mass spectrometric techniques were used to trace the incorporation of [18O]oxygen into metabolites of the photorespiratory pathway. Glycolate, glycine and serine extracted from leaves of the C3 plants, Spinacia oleracea L., Atriplex hastata and Helianthus annuus which were exposed to [18O]oxygen at the CO2 compensation point were heavily labeled with 18O. In each case only 1 of the carboxyl oxygens was labeled. The abundance of 18O in this oxygen of glycolate reached 50-70% of that of the O2 provided after only 5-10 s exposure to [18O]oxygen. Glycine and serine attained the same final enrichment after 40 and 180 s, respectively. This confirms that glycine and serine are synthesized from glycolate. The labeling of photorespiratory intermediates in intact leaves reached a mean of 59% of that of the (2 provided in the feedings. This indicates that at least 59% of the glycolate photorespired is synthesized with the fixation of molecular O2. This estimate is certainly conservative owing to the dilution of labeled O2 at the site of glycolate synthesis by photosynthetic O2. The yield of 18O in glycolate synthesized was examined in vitro by isolated intact spinach chloroplasts in a system which permitted direct sampling of the isotopic composition of the O2 at the site of synthesis. The isotopic enrichment of glycolate from such experiments was 90-95% of that of the O2 present during the incubation. The carboxyl oxygens of 3-phosphoglycerate also became labeled with 18O in 20 and 40 min feedings with [18O] to intact leaves at the CO2 compensation point. Control experiments indicated that this label was probably due to direct synthesis of 3-phosphoglycerate from glycolate during photorespiration. The mean enrichment of 3-phosphoglycerate was 14 .+-. 4% of that of glycine or serine, its precursors of the photorespiratory pathway, in 10 separate feeding experiments. Possibly this constant dilution of label indicates a constant stoichiometric balance between photorespiratory and photosynthetic sources of 3-phosphoglycerate at the CO2 compensation point. O2 uptake sufficient to account for about half of the rate of 18O fixation into glycine in the intact leaves was observed with intact spinach chloroplasts. Oxygen uptake and production by intact leaves at the CO2 compensation point indicate about 1.9 oxygen exchanged per glycolate photorespired. The fixation of molecular O2 into glycolate plus the peroxisomal oxidation of glycolate to glyoxylate and the mitochondrial conversion of glycine to serine can account for up to 1.75 O2 taken up per glycolate. These studies provide new evidence which supports the current formulation of the pathway of photorespiration and its relation to photosynthetic metabolism. The experiments described also suggest new approaches using stable isotope techniques to study the rate of photorespiration and the balance between photorespiration and photosynthesis in vivo.