Glycine Metabolism in Intact Leaves by in Vivo 13C and 15N Labeling

The Journal of Biological Chemistry (2005) 280, 39238-39245

ABSTRACT
Solid-state 13C NMR measurements of intact soybean leaves labeled by 13CO2 (at subambient concentrations) show that excess glycine from the photorespiratory C2 cycle (i.e. glycine not part of the production of glycerate in support of photosynthesis) is either fully decarboxylated or inserted as 13C-labeled glycyl residues in proteins. This 13C incorporation in leaf protein, which is uniformly 15N labeled by 15NH415NO3, occurs as soon as 2 min after the start of 13CO2 labeling. In those leaves with lower levels of available nitrogen (as measured by leaf nitrate and glutamine-glutamate concentrations), the excess glycine is used primarily as glycyl residues in protein.

The photorespiratory pathway (adapted from Ogren, Ref. 3). Oxygenation of 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.

Cross-polarization magic angle spinning 13C NMR spectrum of an intact lyophilized soybean leaf labeled for 6 min with 13CO2 at 300 ppm (by volume). This is a difference spectrum resulting from the subtraction of the spectrum of an unlabeled leaf. Each of the spectra in the difference resulted from the accumulation of 110,000 scans (using a 2-ms matched cross-polarization transfer at 62.5 kHz with a 1.5-s repeat time). The relative scaling of the two spectra was chosen to minimize the natural-abundance difference peaks between 10 and 30 ppm. Only peaks arising from the 13C label remain, and their integration gives an accounting of total 13C assimilation in the leaf during the labeling period. Two carbonyl carbon difference peaks (in blue) are observed: one at 179 ppm (near the chemical shift of the carboxyl carbon of free glycine) and the other at 171 ppm (the characteristic chemical shift of the peptide carbonyl carbon of glycyl residues in alpha-helical local conformations). The 171-ppm peak and a 43-ppm methylene carbon peak (both in red) decrease in intensity following a 13C{15N} dipolar evolution period for 1 ms (insets), indicating directly bonded 13C-15N pairs.

Bottom, cross-polarization magic angle spinning 13C NMR spectrum of an intact lyophilized soybean leaf labeled for 6 min with 13CO2 at 300 ppm (by volume). The carbonyl carbon peak has a maximum at 176 ppm and the shoulder at 171 ppm (most of which is due to natural abundance 13C) has been inverted by a frequency-selective pulse. The difference spectrum at the top results from subtracting the bottom spectrum from the corresponding spectrum with no frequency-selected inversion, both obtained after a 10-ms delay for equilibration of spin populations (stored along the static magnetic field) by 13C-13C spin diffusion. Each of the spectra in the difference resulted from the accumulation of 230,000 scans. The difference spectrum shows the inverted shoulder (and its spinning sidebands), together with a peak at 43 ppm arising from 13C directly bonded to the carbonyl carbon 13C with a shift of 171 ppm. The peak at 43 ppm does not appear in the corresponding difference spectrum of an unlabeled leaf (inset). Both difference spectra have a peak near 25 ppm, which is assigned to various aliphatic carbons in protein side chains proximate to ester or acid carbonyl carbons in pectins, all at natural abundance. Magic angle spinning was at 7143 Hz.

Bottom, cross-polarization magic angle spinning 13C NMR spectrum of an intact lyophilized soybean leaf labeled for 6 min with 13CO2 at 300 ppm (by volume). Only the region of the spectrum between 0 and 90 ppm is shown. The carbonyl carbon shoulder at 181 ppm has been inverted by a frequency selective pulse. The difference spectrum at the top results from subtracting the bottom spectrum from the corresponding spectrum with no frequency selected inversion, both obtained after a 10-ms delay for equilibration of spin populations stored along the static magnetic field by 13C-13C spin diffusion. Each of the spectra in the difference resulted from the accumulation of 230,000 scans. The difference spectrum shows a methylene carbon peak at 43 ppm that is larger than that observed in the corresponding difference spectrum of an unlabeled leaf (inset). The superimposed spectra in red were taken with a 1-ms 13C{15N} dipolar evolution period following the 10-ms period for 13C-13C spin diffusion and preceding acquisition. Diminution of signal intensity indicates the presence of directly bonded 13C-15N pairs.

Cross-polarization magic angle spinning 13C NMR spectrum of intact lyophilized soybean leaves labeled for 4 min with 13CO2 at 200 ppm (top) and 300 ppm (bottom). Both are difference spectra resulting from the subtraction of the corresponding spectra of leaves labeled for 2 min. Each of the spectra of the two differences resulted from the accumulation of 20,000 scans. The difference spectrum of the leaf labeled with 300 ppm of 13CO2 shows no accumulation of 13C label in carbonyl carbons between 2 and 4 min of the start of the labeling period.