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Keywords:

  • Calvin cycle;
  • carboxylase;
  • magic-angle spinning;
  • oxygenase;
  • starch

In Dirks et al. (2012), leaves of Glycine max (soybeans) were labeled by 200 and 600-ppm 13CO2 spiked with 11CO2 and the effects of light intensity and water stress on metabolism were examined using a combination of direct positron imaging and solid-state 13C nuclear magnetic resonance (NMR). The 11C labeling identified photosynthetically active regions of the leaf and the NMR analysis determined total 13C assimilation and the partitioning of label into sugars, starch, amino and organic acids, proteins, and protein precursors. The combination of 11C and 13C labeling led to three conclusions for photosynthetically heterogeneous soybean leaves: (1) transient starch deposition is not the temporary storage of sucrose excluded from a saturated sugar-transport system; (2) peptide synthesis is reduced under high-light, high CO2 conditions; (3) all glycine from the photorespiratory pathway is routed to proteins within photosynthetically active regions when the leaf is water stressed and under high-light and low CO2 conditions.

In the preceding Letter in this issue of New Phytologist, Tcherkez (pp. 334–338) covers many topics and makes many claims; the one that directly addresses Dirks et al. (2012) is a challenge to the third conclusion. Tcherkez maintains that the analysis leading to this conclusion was flawed because (1) 11C data for the net carbon assimilation were not given; (2) the solid-state NMR spectra were poorly resolved and contributions from amino and organic acids were neglected; (3) the calculations of decarboxylation rates were inconsistent with the accepted notions of net carbon assimilation near the compensation point, and with the necessity of glycine[RIGHTWARDS ARROW]serine[RIGHTWARDS ARROW]3-phosphoglycerate (3-PGA) to provide needed nitrogen by NH2-recovery from serine.

We respond to each of these assertions below.

Response to assertions by Tcherkez (2013, this issue)

  1. Top of page
  2. Response to assertions by Tcherkez (, this issue)
  3. Perspective
  4. References

11C data

In fact, 11C assimilation data for all experiments examining the effects of CO2 concentration, light intensity, and water stress are given in Dirks et al. (2012). Figure 4 of that paper allows direct comparisons of 11C assimilation (light and CO2 levels as variables) for the four 13C NMR spectra of figure 5. Figures S4 and S5 of the Supporting Information (SI) provide the 11C data for the 200 and 600-ppm CO2 labeling comparison of carbon assimilation under water stress, with the 13C NMR spectra appearing in figures 7 and 8 of the main text. In addition, unlike the situation for 13C solution-state NMR, the total integrated intensities of the 13C solid-state NMR label-only spectra are themselves direct measures of net carbon assimilation. For example, the total integral of figure 5(a) is about three times that of figure 5(b), consistent with the corresponding rates of carbon assimilation in figure 4 (top left and top right, respectively). The y-axis amplitude scales of the four panels of figure 5 of the main text can be directly compared. (Note that the y-axis amplitudes have been expanded by ×2 for the low-light spectra of the panels on the right.) The y-axis amplitudes of the spectra of figures 7 and 8 are related by a scale bar within each figure.

NMR resolution

The assignments of the solid-state NMR spectra of figures 5–8 (Dirks et al., 2012) were made with the help of 13C{15N} rotational-echo double-resonance (REDOR) spectra (Gullion & Schaefer, 1989) in the SI (Dirks et al., 2012, figure S8), and reproduced here as Fig. 1. The leaf of Fig. 1 has been uniformly 15N-labeled to an enrichment of c. 50%. The label-only spectrum Fig. 1 (left, bottom) is similar to that shown in figure 5(c) of Dirks et al. (2012). Both leaves were well watered, under high light and 200-ppm 13CO2. It seems reasonable to suppose that the two leaves were similarly labeled. Using 13C{15N} REDOR, we prove that there is no significant overlap from other compounds at 171 ppm (Fig. 1, top left). This chemical shift is, in fact, recognized as a marker for glycyl residues in peptide bonds (Yu et al., 2010). There is a strong REDOR difference signal at 171 ppm for the leaf labeled by 200-ppm 13CO2 but not for the leaf labeled by 600-ppm 13CO2 (Fig. 1, top right), consistent with the claim in Dirks et al. (2012) that protein synthesis was suppressed in the leaf of figure 5(a) (see red arrow) although possibly not entirely eliminated.

image

Figure 1. Detection of 13C–15N peptide bonds by solid-state NMR. 125-MHz 16-rotor-period 13C{15N} REDOR spectra are shown of two soybean (Glycine max) cultivars labeled for 1 h by 13CO2 at 200 ppm (left) and 600 ppm (right) under full sunlight. No 11C labeling was involved. Natural-abundance backgrounds have been subtracted so the spectra arise from 13C label only. Full-echo spectra are shown at the bottom of the figure and REDOR differences at the top. The inserts show expansions of the 170–180 ppm regions of the spectra. The absolute vertical scales on the left have been increased relative to those on the right by about a factor of two. Magic-angle spinning was at 7143 Hz.

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REDOR also identifies the Cα carbons which are directly bonded to 15N. The glycyl Cα has a unique shift at 42 ppm. The other Cα carbons are in a broad peak centered near 55 ppm. Cegelski & Schaefer (2005) used 13C-13C recoupling to show that most of the glycyl carbons responsible for the 42-ppm REDOR difference peak in leaf spectra like these are directly bonded to the peptide carbonyl carbons responsible for the 171-ppm peak. That is, both signals arise from glycyl residues in proteins or protein precursors. Yu et al. (2010) used frequency-selective REDOR to show that many of the labeled glycyl residues had glycyl residues as nearest neighbors. That is, the labeled proteins had high concentrations of Gly–Gly sequences for labeling with 200-ppm 13CO2. These results are direct experimental NMR facts and require no models or assumptions.

The 55-ppm Cα peak in the REDOR spectrum of Fig. 1 (top left) clearly shows that amino acids other than glycine are present. Thus, the gly[RIGHTWARDS ARROW]ser conversion has likely occurred as well as amino-acid synthesis from labeled sugars. The relative decarboxylation rate (D) of table 1 of Dirks et al. (2012) is therefore obviously not zero (D = 0 would have indicated a completely open C2 pathway). Indeed, the full-echo (S0) carboxyl-carbon peak at 178 ppm is about the same size as the 171-ppm peak and so glycine incorporation into protein at 200-ppm CO2 (full light, no water stress; see also figure 5, lower left, Dirks et al., 2012) can be no > c. 50%. This value is consistent with the labeled sucrose anomeric-carbon peaks (doublet at 100 ppm) which are each about three times as intense as the 171-ppm peptide peak. The latter and one of the 100-ppm anomeric-carbon peaks represent single labeled carbons of protein and sugar repeat units, respectively. For 200-ppm CO2, low light, and no water stress, the 178-ppm carboxyl-carbon peak is reduced (Dirks et al., 2012, figure 5, lower right) as is the sucrose doublet, so glycine incorporation into protein is increased. However, in none of the discussions of glycine metabolism in well-watered leaves cited in the last two paragraphs was the claim made that the decarboxylation rate of glycine is zero and that glycine is routed exclusively to protein.

Decarboxylation rates and water stress

The 11C images and 13C NMR spectra for 200-ppm CO2 labeling under high light and water stress (Dirks et al., 2012, figure 8) are reproduced here in Fig. 2. The label-only spectra (top) are qualitatively different from those of hydrated leaves under high light shown in Fig. 1 of this letter, or figure 5 of Dirks et al. (2012). Now the labeled carbonyl-carbon peak (centered near 171 ppm) and the glycyl Cα peak (42 ppm) are several times more intense than the sucrose doublet (100 ppm). This result is unambiguous even though resolution of the spectrum is poor. Carbon assimilation at low CO2 is readily measured by the integral of the entire spectrum. The net carbon assimilation for the most photosynthetically active region at 200-ppm CO2 (Fig. 2, upper right) is less than that measured for 600-ppm CO2 labeling by a factor of 2.2 (Dirks et al., 2012). The net carbon assimilation for an average over the 11C imaged part of the leaf is less by a factor of 3 (Dirks et al., 2012, figures 7 and 8), and for the whole leaf, less by a factor of 4.6. The latter was determined by the 11C uptake for 2 min (Dirks et al., 2012, figures S4, S5, SI). The 11C images and 13C NMR spectra for 600-ppm CO2 labeling under high-light and water stress show strong sugar and starch peaks but no peptide peak for the most photosynthetically active region of the leaf (Dirks et al., 2012, figure 7, upper right).

image

Figure 2. Use of a template from a 11C image to identify by NMR photosynthetically active regions of a water-stressed soybean (Glycine max) leaf in a low CO2 atmosphere. (a) NMR spectra of three fragments of a lyophilized leaf from a combined 11C and 13C labeling experiment using 200-ppm 13CO2 and high-light conditions (710 μmol photon m−2 s−1). The presentation has the same format as used in figure 6 (Dirks et al., 2012). The red and black cross-hatching (top left) indicate carbon partitioning into carbohydrate and protein, respectively. The inset (top middle) shows the deconvolution of the 170–180 ppm chemical-shift region into carboxyl and peptide carbonyl carbons by using shift positions and line shapes determined by rotational-echo double resonance (Dirks et al., 2012, Supporting Information, figure S8). The dotted red and blue lines are for internal peak-height comparisons. An absolute vertical-scale intensity bar is shown in the bottom panel (extreme right) for comparison to intensities in figure 7 (Dirks et al., 2012) where the scale has been compressed by a factor of 2.3. (b) Movement of photosynthate in the leaf shown in the same format as that used in figure 3 (Dirks et al., 2012).All of the positron images involved 10 min of signal averaging and have been corrected for radioactive decay.

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Making simple assumptions about the relative carboxylase and oxygenase rates for the most photosynthetically active region of the leaf of Fig. 2 (top right), we estimated that the ratio of net carbon assimilation at 600-ppm CO2 to that at 200-ppm CO2 was 4.6 (D ≈ 1) or 2.6 (D ≈ 0) (Dirks et al., 2012, table 1). The experimental value for this region is 2.2. Thus, it appears that all glycine from the photorespiratory pathway is routed to proteins within the photosynthetically active region when the leaf is water stressed and under high light and low CO2 conditions. We reached this conclusion by comparison of total 13C integrated intensities and not by an attempt to measure peak intensities of poorly resolved spectra like those in Fig. 2. The claim that D ≈ 0 was not made for all regions of the imaged leaf, or for the average of the entire leaf. In addition, the caveat stated in Dirks et al. (2012) is still true: a completely open C2 pathway is not possible for long periods for any part of the leaf, or for any period for a nitrogen-starved leaf. In the latter situation, none of the glycine from the oxygenase pathway is routed to protein (see Dirks et al., 2012, figure S10, SI,) and all glycine goes to serine and is returned to the Calvin cycle via 3-PGA.

Perspective

  1. Top of page
  2. Response to assertions by Tcherkez (, this issue)
  3. Perspective
  4. References

Whether all the glycine from the C2 pathway in a water-stressed photosynthetically active region in high light and 200-ppm CO2 goes to protein or only 50% goes to protein, the metabolically important biological facts are first, that the percentage of glycine going directly to protein in this situation is large, and second, that for the same photosynthetically active region under 600-ppm CO2, none of the glycine goes to protein. There are C3 plants which route glycine to protein at 600-ppm CO2 to the same extent as at 200-ppm CO2 (Yu et al., 2010). Soybean is not one of them. The implications of the soybean response to high CO2 was a major focus of the work reported in Dirks et al. (2012).

References

  1. Top of page
  2. Response to assertions by Tcherkez (, this issue)
  3. Perspective
  4. References