NMR determination of photorespiration in intact leaves using in vivo 13CO2 labeling

Journal of Magnetic Resonance Volume 178, Issue 1, January 2006, Pages 1-10

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.

The photorespiratory pathway (adapted from W.L. Ogren, Photorespiration: pathways, regulation, and modification, Ann. Rev. Plant Physiol. 35 (1984), pp. 415--442). Oxygenation of ribulose bisphosphate (RuBP) leads to the production of the 2-carbon phosphoglycolate (red), which is partially recycled by serine synthesis leading to glycerate. This process requires machinery distributed over three organelles: the chloroplast (C), the peroxisome (P), and the mitochondrion (M). Concurrent energy balance processes via NAD/NADH and ATP/ADP conversions are omitted. The numbers in parentheses indicate stoichiometry.

The 13CO2 labeling of a soybean leaf using a compact-disc case as a labeling chamber. The labeling gas (21% O2, either 200 or 300-ppm 13CO2, and the balance N2) entered at the bottom left through a copper pipe closed at the end and with multiple exit holes along the sides. At the end of the labeling period, the leaf was cut from its stem, immersed in liquid nitrogen, and subsequently lyophilized.

13C{31P} eight rotor-cycle rotational-echo double-resonance (REDOR) spectra of soybean leaves labeled by 300-ppm (by volume) 13CO2 for 2--6 min. The REDOR difference spectra (S0 - S) are at the top of the figure and the full-echo spectra (S0) at the bottom. The full-echo spectra have been normalized by their natural-abundance methyl-carbon peaks (dotted lines). The REDOR difference arises from Calvin-cycle intermediates with 13C-labeled phosphorylated carbons. Each spectrum resulted from the accumulation of 50,000 scans. Magic angle spinning was at 7143 Hz.

Cross-polarization magic-angle 13C NMR spectra of intact lyophilized soybean leaves labeled for 2, 4, or 6 min with 13CO2 at 200 ppm (bottom) or 300 ppm (top), both concentrations by volume. The 62.5-kHz matched spin-lock contact time was 1 ms. Each spectrum resulted from the accumulation of 20,000 scans. The accumulation of label (dotted lines) is approximately linear between 2 and 6 min at both labeling concentrations. These spectra were used in the determination of the carbon assimilation rates.

Cross-polarization magic-angle 13C NMR spectrum of label accumulated by a soybean leaf labeled for 2 min with 13CO2 at 400 ppm (by volume), and compared with leaves labeled with 13CO2 at 300 and 200 ppm (inset). These are difference spectra resulting from the subtraction of the spectrum of an unlabeled leaf. Each of the spectra of the differences resulted from the accumulation of 130,000 scans. The inset (in blue) shows that the distribution of label between the 171- and 179-ppm carbonyl-carbon peaks changes as a function of the concentration of 13CO2 used for labeling.

At reduced external CO2, the relative rates of flow of 13C (shaded) and 12C (white) into the Calvin cycle are unchanged. The ratio of photosynthetic CO2 (in) to photorespiratory CO2 (out) is also unchanged, but the flow of carbon into glycine has increased. Part of this glycine is converted into glycerate, while the remainder may be incorporated into other products, or, depending on the metabolic state of the leaf, decarboxylated.