NMR determination of photorespiration in intact leaves using in vivo 13CO2 labeling.
Solid-state 13C NMR measurements of intact soybean leaves labeled by 13CO2 lead to the conclusion that photorespiration is 17% of photosynthesis for a well-watered and fertilized plant. This is the first direct assessment of the level of photorespiration in a functioning plant. A 13C{31P} rotational-echo double-resonance (REDOR) measurement tracked the incorporation of 13C label into intermediates in the Calvin cycle as a function of time. For labeling times of 5 min or less, the isotopic enrichment of the Calvin cycle depends on the flux of labeled carbon from 13CO2, relative to the flux of unlabeled carbon from glycerate returned from the photorespiratory cycle. Comparisons of these two rates for a fixed value of the 13CO2 concentration indicate that the ratio of the rate of photosynthesis to the rate of photorespiration of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in soybean leaves is 5.7. This translates into a photorespiratory CO2 loss that is 21% of net CO2 assimilation, about 80% of the value estimated from Rubisco kinetics parameters. The ratio of rates is reduced at low external CO2 concentrations, as measured by net carbon assimilation rates. The carbon assimilation was determined from 13C-label spin counts converted into total carbon by the REDOR-determined isotopic enrichments of the Calvin cycle. The net carbon assimilation rates indicate that the rate of decarboxylation of glycine is not directly proportional to the oxygenase activity of Rubisco as is commonly assumed.