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. 2012 Oct 9;2:719. doi: 10.1038/srep00719

Tracking Stable Isotope Enrichment in Tree Seedlings with Solid-State NMR Spectroscopy

Charlotte E Norris 1,a, Sylvie A Quideau 1, Simon M Landhäusser 1, Guy M Bernard 2, Roderick E Wasylishen 2
PMCID: PMC3466447  PMID: 23056911

Abstract

Enriching plant tissues with 13C and 15N isotopes has provided long-lasting, non-reactive tracers to quantify rates of terrestrial elemental fluxes (e.g., soil organic matter decomposition). However, the molecular location and level of isotope enrichment may differ among plant tissues. This factor is central to the integrity and interpretation of tracer data, but is seldom considered in experiments. We propose a rapid, non-destructive method to quantify molecular isotope allocation using solid-state 13C and 15N nuclear magnetic resonance spectroscopy. With this method, we tracked and quantified the fate of multiple pulses of 13CO2(g) and K 15NO3(l) in boreal tree seedling roots and leaves as a function of time. Results show that initial preferential 13C carbohydrate enrichment in the leaves was followed by redistribution to more complex compounds after seven days. While 13C allocation within the roots was uniform across molecules, 15N results indicate an initial enrichment of amine molecules after two hours.

Terrestrial ecosystems are changing due to anthropogenic pressures, leading to far-reaching effects on global biogeochemical cycles. Assessing the net CO2 flux between terrestrial ecosystems and the atmosphere remains one of the largest uncertainties in understanding of the global carbon cycle. Mechanisms controlling the persistence of soil organic matter are complex and reflect the interacting influences of abiotic conditions, nutrient supply, microbial community structure, and organic matter composition1. Soils globally contain twice as much carbon as the atmosphere and world's vegetation combined, yet how this major carbon pool will respond to global environmental change is not well understood. Carbon storage in boreal forest soils, which alone represents close to 25% of the global soil carbon stocks, is particularly susceptible as northern regions are anticipated to experience warming of up to 5°C by 2100 due to climate change2.

Accurate and quantitative rates of soil carbon fluxes are increasingly required to better constrain predictions of soil carbon turnover. Carbon transfer from plant residues to soils occurs both as surface litterfall or below-ground via root exudates and turnover. Carbon-13 (natural abundance 1.07%) and 15N (natural abundance 0.364%) have been used to track both of these elements as they are mineralized from the decomposing enriched plant materials, taken up by the soil microbial biomass, stabilized into organic matter, or alternatively respired back to the atmosphere3,4. Vascular plants, particularly woody perennial species, contain a range of complex tissues made up of varying amounts of different plant biopolymers5,6 depending on environmental conditions7. In addition these biopolymers display different decay rates, at least initially, with compounds such as lignins and waxes taking longer to decompose in the soil environment compared to proteins, soluble sugars, or cellulose8. Consequently, molecular characterization of labeled litter inputs should be combined with isotopic flux measurements if the fluxes are to be accurately interpreted in the broader context of ecosystem biogeochemical cycling.

Historically, isotope labeling of plant material originated with the use of radioactive carbon (14C)9,10. With increased availability of stable isotopes (notably 13C and 15N) and improved quantitative analytical instrumentation, stable isotopes have become an important tool for tracer studies in terrestrial ecosystems11. Experimentally, enrichment is either achieved by continuous addition of the stable isotope or by applying pulses. Since the introduction of pulse chase labeling9, the method has been adopted as the technique of choice compared to the more cumbersome continuous labeling apparatus. Numerous studies have used stable isotope enriched organic matter to examine carbon12,13,14 and nitrogen biogeochemical cycles12. However, for methodological reasons, most studies have been restricted to non-woody fast growing graminoids that show little complexity in tissues and their associated biopolymers. Here, we selected trembling aspen (Populus tremuloides Michx.), a dominant upland tree species in North-American boreal forests15, which is closely related to the equally common Populus tremula in the northern hemisphere of Eurasia. Aspen leaf tissues display a diverse and complex composition including cellulosic, aromatic and alkyl carbon species5. The objective of the present study was to determine how pulse-labeling with13CO2(g) and K 15NO3(l) affected isotopic enrichment in aspen seedlings at both the elemental and biopolymer level. To provide the greatest contrast in tissue composition, we present results on both foliage and woody root tissues for isotopic (13C, 15N) abundance in response to three pulses and two chase periods (two hours and seven days). In addition, we quantified and characterized isotope assimilation in plant biopolymers using solid-state 13C and 15N nuclear magnetic resonance (NMR) analysis.

Results

The 13C and 15N concentrations increased in the seedling tissues with each successive pulse label; however, timings and patterns of isotope enrichment were different (Figure 1). Two hours after the first 13CO2 pulse, leaf tissues had doubled in 13C concentrations compared to background levels. Although not all 13C was retained within the leaves, a steady enrichment to 2.7 atom % from a background concentration of 1.1 atom % was observed with successive pulses. Root tissues did not show any detectable 13C enrichment two hours after the first pulse, but a significant increase in 13C concentration was evident seven days later (1.4 atom %). In contrast to 13C enrichment patterns, 15N enrichment was first noted in the roots two hours after the first pulse, with subsequent labels further increasing the concentration from 0.4 to 10.7 atom % 15N. Enrichment of 15N in the leaves could be detected seven days after the first pulse label. Final concentrations of 13C and 15N for unlabeled leaves and roots were similar to the initial ones; i.e., without pulse-labeling the proportion of 13C to 12C and 15N to14N did not change during the experiment.

Figure 1. Atom percent concentration of 13C and 15N in seedling leaves and roots at different pulse and chase times.

Figure 1

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Chase time of control, 2 hour or 7 day is indicated by shading in the box. The number of pulses (0–3) is indicated by a number at the base of the barplot. Error bars represent one standard deviation (n = 6).

Variation in the biopolymer allocation of 13C in aspen seedlings with repeated pulses and multiple chase times depended on the tissue type (Figure 2). As can be seen on the 13C spectra of leaves, the O-alkyl signal (corresponding to oxygen-bonded or carbohydrate type carbons) was strongest two hours following labeling, and corresponded to 41–48% of the total integrated spectral area (Figure 3). In comparison, the alkyl carbon signal (from aliphatic carbons in polymethylene such as cutin) was originally much smaller, but increased with time, so that the O-alkyl and alkyl peaks were closer in intensity with a longer (seven days) chase period (Figure 2). The resolution of peaks attributed to aromatic and phenolic carbons (arising from lignin and tannins) improved with 13C enrichment, and the intensity of both increased in proportion with the longer chase time.

Figure 2. Direct polarization 13C NMR spectra with control-initial, one pulse and 2 hour chase and one pulse 7 day chase 13C enriched seedling leaves and roots.

Figure 2

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Common integral regions representing the different magnetic environments of carbon are shown on the spectra.

Figure 3. Relating the proportion of O-alkyl carbon to 13C enrichment for the multi-pulse and multi-chase stable isotope labeling of seedling leaves and roots.

Figure 3

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One standard deviation for O-alkyl carbon (n = 2) and carbon-13 atom % (n = 6) are indicated by error bars on the graph.

Allocation of 15N to plant biopolymer groups, as with 13C, varied depending on the chase time (Figure 4). Nitrogen characterization was limited to a subset of labeled samples as the signal from nuclei other than amides was indistinguishable from the noise on the control samples with natural abundance 15N levels. Results are qualitative as amine nitrogen is known to be under-represented, and nonprotonated nitrogen is difficult to dectect16. After two pulses and a chase time of 7 days, the 15N NMR spectra for leaves and roots both had NMR peaks characteristic of amide and amine nitrogen well above the background noise (Figure 4).

Figure 4. Cross polarization 15N NMR spectra of seedling leaves and roots indicating qualitative differences between the two chase times (2 hour and 7 day).

Figure 4

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The 7 day chase samples had experienced 2 pulses while the 2 hour samples had 3 pulse of 13CO2. Common integral regions representing the different magnetic environments of nitrogen are shown on the spectra. The asterisks indicate spinning side bands.

Discussion

Solid-state NMR spectroscopy may be used as an analytical tool to characterize the environment surrounding nuclei of interest (e.g., 13C or 15N) from which the chemical composition of plant or soil organic matter may be identified5,17. The magic angle spinning technique (MAS) entails spinning samples rapidly about the magic angle (54.7°) relative to the applied magnetic field. The method greatly reduces the effects of chemical shift anisotropy, resulting in significant improvements in resolution compared to what is obtained for solid-state NMR spectra of stationary samples. The method also reduces the effects of homonuclear (e.g., 13C-13C) dipolar coupling. This technique is often combined with cross polarization (CPMAS) whereby the magnetization is transferred from abundant spins (usually 1H) to less abundant spins; i.e., 13C or 15N. The latter technique provides a signal enhancement as great as the ratio of the magnetogyric ratios of the nuclei, ≈4 for 13C and ≈10 for 15N. However, since in most cases the less abundant spins also have a much longer spin-lattice relaxation time (T1) then do the 1H nuclei, the greatest benefit of the CP technique is usually that the repetition time of the experiment is controlled by the T1 value of the latter. Hence, one may acquire many more transients in a given period of time, greatly improving the signal/noise ratios of the spectra18. However, it is important to realize that spectra obtained with CPMAS are not quantitative, since the signal arising from the nuclei are not all enhanced to the same extent. An alternative to the CPMAS technique is to obtain spectra with MAS using direct polarization (DP), which provides qualitative results provided the recycle time is significantly greater than the greatest T1 value for the nuclei of interest19. For the samples in the present study, 13C spectra were obtained by DP; to eliminate the interference from natural abundance 13C in either the probe or the rotor drive tips and end caps, a modified DP spin-echo pulse sequence was used20. The 15N enrichment was qualitatively characterized by the CPMAS technique since obtaining suitable 15N DP spectra was not practical.

Chemistry of seedling roots was different from leaves in that the samples did not exhibit much variation with the different levels of enrichment (Figures 2 and 3). Hence, the roots showed fairly uniform 13C enrichment across the different biopolymers. For the leaves, the shift in intensity of the O-alkyl carbon peak between the two chase times likely reflects the initial fixation of carbon as glucose followed by a partial redistribution to more complex compounds such as cutin or lignin. On the other hand, uniform 13C enrichment within the root biopolymers reflects non-discriminatory incorporation into new structural root tissues, including cellulose and lignin, rather than preferential accumulation into O-alkyl C in the form of soluble sugars or starch reserves. While the 15N NMR spectra reflected the acquisition and allocation of nitrate by the seedling; two hours after the K 15NO3 pulse, roots had taken up and reduced the labelled nitrate into amine groups. Roots contain nitrogen reductase and other enzymes for amino acid and amide formation, which allows the roots to synthesize organic solutes for nitrogen distribution throughout the plant21,22. Our results support this pathway as evidenced by the relatively higher amide/amine peak ratio on the root spectra after a 7 day chase period (Figure 4).

For biogeochemical studies aimed at elucidating organic matter processes and ecosystem fluxes, a distinction between plant-labeled material and native soil organic matter needs to be made. Hence knowledge of the isotope enrichment levels across different tissues and biopolymers is essential. Here we demonstrated that both the allocation of 13C and 15N in leaves and roots was dependent on the chase time (Figure 1). Similar results have been reported during pulse labeling of birch (Betula papyrifera) and fir (Pseudotsuga menziesii) trees, where the initial increase in foliage 13C concentration decreased while that of roots increased with a longer chase time23. The level of enrichment also needs to be sufficient for planned biogeochemical tracer studies. Enrichment of 13C at the end of our labeling experiment was 2.7% in leaves and 1.4 atom % in roots. This is comparable to levels previously reported for needles (1.6% 13C) and roots (1.4 atom % 13C) of Douglas-fir seedlings, which were demonstrated to be sufficient to follow tracers during a soil incubation study24. The 15N enrichment levels for leaves (8.7% 15N) and roots (10.7 atom % 15N) also were comparable to enriched ponderosa pine tissues used in a field decomposition study25. The NMR spin-active isotopes of carbon (13C) and nitrogen ( 15N) make up 1.1 and 0.4%, respectively of the total number of atoms at natural abundance levels in our control samples. With tissue enrichment, there was a subsequent increase in the abundance of both spin-1/2 atoms, which directly translated into an enhanced signal-to-noise ratio and improved characterization. Using this technique, we were able to identify that uniform labeling required a one-week chase period for both the leaves and the roots. Combining NMR characterization with stable isotopic labeling has been successfully applied in other fields, notably plant metabolomics26, but, to our knowledge, this represents the first use of this rapid, non-destructive method to quantify molecular isotope allocation using DP 13C NMR and 15N CPMAS spectroscopy for stable and non-reactive tracers in biogeochemical studies. Further work using multi-dimensional NMR methods or a combined instrument approach (e.g., solid-state NMR, solution-state NMR, compound specific isotope analysis with isotope ratio mass spectrometry), as in the area of plant metabolomics26, would continue to refine our knowledge on the allocation of the stable isotopes within the tissues. With knowledge of these methods not only could enriched plant organic matter for decomposition in biogeochemical studies be better characterized but also investigations on the abiotic influences (e.g., water stress) or biotic interactions (e.g., insect grazing) on root exudates and plant physiology could be elucidated.

Methods

Seedling growth and enrichment

Thirty three one-year-old aspen seedlings, grown from an open pollinated aspen seed source at a commercial nursery (Smoky Lake Nursery, Smoky Lake, Alberta), were used in this study. Dormant seedlings were planted into pots (15 cm wide and 17 cm high) filled with a mixture of two thirds peat and one third sand. Seedlings were grown in a greenhouse with an average air temperature of 21°C, a photoperiod of 16 hours, and daily watering. Seedlings flushed after 7 days and seedlings were fertilized once with a liquid application of 2.0 g L−1 of 20-20-20 N-P-K with chelated micronutrients (Plant Products Co., Brampton, Ontario). After 5 weeks of growth, three seedlings were harvested to establish initial baseline measurements and three additional seedlings were transferred to a separate greenhouse with identical growing conditions to provide the source for the final, unenriched (control) measurements.

Seedlings were labeled once per week for three consecutive weeks. Following each pulse (label), harvesting occurred after two distinct chase periods: two hours and seven days after the pulse (except for the third pulse). Labeled nitrogen ( 15N) was added to the seedlings via an application of 50 ml K 15NO3 (0.207 M of N, 60 atom % 15N) solution (Sigma-Aldrich Chemicals, St. Louis, MO, USA). Enrichment of carbon (13C) was achieved by adding 100 ml of 99.9% 13C CO2 (Cambridge Isotope Laboratories, Inc. Andover, MA, USA) to each seedling enveloped in a Mylar tubular bag (36 cm diameter with the length varying depending on the seedling height (VacPac®, Baltimore MD). A 50 ml gastight syringe (Hamilton, Reno NV) was used to inject 13CO2 gas. Seedlings were allowed to take up the 13CO2 gas for 30 minutes before the bags were removed. At sampling time (two hours and seven days after the pulse), three seedlings were randomly selected for harvesting and separated into three components: leaves, stem, and roots. Plant components were oven dried at 60°C, weighed, coarsely ground and stored until further analysis. The complete aspen seedling enrichment experiment was run twice.

Laboratory analyses

Samples were finely ground using a Retsch MM200 ball mill grinder (Retsch Inc. Newtown, USA) and analysed for total carbon and nitrogen contents on a Costech ECS 4010 Elemental Analyzer (Costech Analytical Technologies Inc. Valencia, USA). The isotopic composition of the samples was measured on a Costech ECS 4010 Elemental Analyzer equipped with a thermocouple detector joined to a Finnigan Deltaplus Advantage Isotopic Ratio Mass Spectrometer (ThermoFinnigan, Bremen, Germany).

Nuclear magnetic resonance

Samples for 13C were analyzed on a Chemagnetics CMX Infinity 200 [magnetic field (B0) = 4.70 T, Larmor frequency (νL) (13C) = 50.3 MHz] spectrometer with a 7.5 mm double-resonance MAS probe capable of high-power 1H decoupling (Varian, Ft. Collins, CO, USA). Samples were loaded into a 7.5 mm OD zirconium oxide rotor with Kel-F drive tips and end caps, and spacers made of Teflon (DuPont, Circleville, OH, USA). The magic angle was set to 54.74° by maximizing the 79Br NMR signal for spinning sidebands of KBr. Carbon-13 spectra were referenced to tetramethylsilane (0 ppm) by setting the high-frequency adamantane peak to 38.56 ppm27.

Direct polarization 13C with a spin echo pulse (90 - τ - 180 - τ -ACQ- τ = 400 s) was used to acquire spectra18,20. A total of 500 transients (2000 for the control initial leaves) were acquired for each free induction decay (FID) with: 5.0 kHz spinning rate, 4.5 µs 90° pulse width, and 100 s recycle time. Processing of the resulting FID was performed with WIN-NMR version 6.0 (Bruker, Germany). Processing the spectra included zero filling to 1 K, line broadening of 100 Hz with phase and baseline correction followed by integration of the spectra. Previously published descriptions on peak identification and classification of integral regions were used to separate the following six regions5,8: alkyl (0–47 ppm), O-alkyl (47–93 ppm), di-O-alkyl (93–112 ppm), aromatic (112–140 ppm), phenolic (140–165 ppm), and carbonyl (165–185 ppm).

Nitrogen-15 NMR spectra of magic angle spinning samples were acquired on a Bruker Avance 500 NMR spectrometer, operating at 50.69 MHz for 15N. Spectra were acquired with ramped CP, a 4.0 μs 1H π/2 pulse, a 1 ms contact time and a recycle delay of 3.0 s. Samples were spun at 6 kHz. Spectra were obtained by adding 8 000 to 20 000 transients and were referenced such that δiso = 0 for the isotropic 15N resonance of methyl nitrate by setting the 15N isotropic peak of glycine- 15N (98% 15N) to −347.54 ppm28. The latter sample was used to optimize acquisition parameters. Spectra of 15N enriched samples contained 4 distinct regions attributed to amide (−220 to −288 ppm), arginine sidechain (−288 to −324 ppm) and amine (−324 to −365 ppm) nitrogen sites16; the much more intense signal from the amide site contained spinning sidebands whose integrated intensities were added to that of the isotropic peak. In addition, to confirm the 13C spectra acquired on the CMX 200, select samples were analyzed with either ramped CP or Bloch decay pulse sequences on the Avance 500 spectrometer operating at a frequency of 125.81 MHz. for 13C.

Statistics

Descriptive statistics, sample means and standard deviations were determined using R version 2.11.0 (The R Foundation for Statistical Computing).

Author Contributions

C.E.N. designed and conducted the experiment, processed the data and prepared the manuscript. S.A.Q. was instrumental in the project design, supervision and manuscript preparation. S.M.L. was involved in project design and provided seedlings, greenhouse space and tree growth expertise. G.M.B. acquired 15N spectra and, with R.E.W., provided both expertise and NMR time.

Acknowledgments

Direct financial support was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Oil Sands Network for Research and Development through grants to R.E.W., S.A.Q. and S.M.L. R.E.W. also acknowledges the Government of Canada for a Canada Research Chair in Physical Chemistry.

References

  1. Schmidt M. W. I. et al. Persistence of soil organic matter as an ecosystem property. Nature 478, 49–56 (2011). [DOI] [PubMed] [Google Scholar]
  2. Christensen J. H. et al. in Climate Change 2007: The Physical Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change (eds. Solomon S., et al.) 847–892 (Cambridge University Press, Cambridge, 2007). [Google Scholar]
  3. Swanston C. W. & Myrold D. D. Incorporation of nitrogen from decomposing red alder leaves into plants and soil of a recent clearcut in Oregon. Can. J. For. Res. 27, 1496–1502 (1997). [Google Scholar]
  4. Moore-Kucera J. & Dick R. P. Application of 13C-labeled litter and root materials for in situ decomposition studies using phospholipid fatty acids. Soil Biol. Biochem 40, 2485–2493 (2008). [Google Scholar]
  5. Preston C. M. & Trofymow J. A. Canadian Intersite Decomposition Experiment Working Group. Variability in litter quality and its relationship to litter decay in Canadian forests. Can. J. Bot. 78, 1269–1287 (2000). [Google Scholar]
  6. Hancock J. E. et al. Plant growth, biomass partitioning and soil carbon formation in response to altered lignin biosynthesis in Populus tremuloides. New Phytol 173, 732–742 (2007). [DOI] [PubMed] [Google Scholar]
  7. Donaldson J. R., Kruger E. L. & Lindroth R. L. Competition- and resource-mediated tradeoffs between growth and defensive chemistry in trembling aspen (Populus tremuloides). New Phytol 169, 561–570 (2006). [DOI] [PubMed] [Google Scholar]
  8. Kögel-Knabner I. The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter. Soil Biol. Biochem 34, 139–162 (2002). [Google Scholar]
  9. Svejcar T. J., Boutton T. W. & Trent J. D. Asessment of carbon allocation with stable carbon isotope labeling. Agron. J. 82, 18–21 (1990). [Google Scholar]
  10. Warembourg F. R. & Kummerow J. in Carbon Isotope Techniques. (eds. Coleman D. C., & Fry B.) 11–37 (Academic Press, Inc., San Diego, 1991).
  11. Dawson T. E., Mambelli S., Plamboeck A. H., Templer P. H. & Tu K. P. Stable isotopes in plant ecology. Annu. Rev. Ecol. Syst. 33, 507–559 (2002). [Google Scholar]
  12. Bird J. A., van Kessel C. & Horwath W. R. Stabilization of 13C-carbon and immobilization of 15N-nitrogen from rice straw in humic fractions. Soil Sci. Soc. Am. J. 67, 806–816 (2003). [Google Scholar]
  13. Bottner P. et al. Decomposition of 13C-labelled plant material in a European 65–40° latitudinal transect of coniferous forest soils: simulation of climate change by translocation of soils. Soil Biol. Biochem 32, 527–543 (2000). [Google Scholar]
  14. Arevalo C. B. M., Bhatti J. S., Chang S. X. & Sidders D. Distribution of recent photosynthates in saplings of two hybrid poplar clones. Comm. Soil Sci. Plant Anal 41, 1004–1015 (2010). [Google Scholar]
  15. Little Jr E. L. Atlas of Unites States trees (U.S. Dept. of Ag. For. Serv., Washington DC, 1971). [Google Scholar]
  16. Smernik R. J. & Baldock J. A. Solid-state 15N NMR analysis of highly 15N-enriched plant materials. Plant Soil 275, 271–283 (2005). [Google Scholar]
  17. Norris C. E., Quideau S. A., Bhatti J. S. & Wasylishen R. E. Soil carbon stabilization in jack pine stands along the Boreal Forest Transect Case Study. Glob. Change Biol 17, 480–494 (2011). [Google Scholar]
  18. Bryce D. L. et al. Practical aspects of modern routine solid-state multinuclear magnetic resonance spectroscopy: one-dimensional experiments. Can. J. Anal. Sci. Spectros 46, 46–82 (2001). [Google Scholar]
  19. Simpson M. J. & Preston C. M. in Soil Sampling and Methods of Analysis. (eds. Carter M. R., & Gregorich E. G.) 681–692 (CRC Press, Boca Raton USA, 2007).
  20. Smernik R. J. & Oades J. M. Background signal in solid state 13C NMR spectra of soil organic matter (SOM)- quantification and minimization. Solid State Nucl. Magn. Reson 20, 74–84 (2001). [DOI] [PubMed] [Google Scholar]
  21. Pate J. S. Uptake, assimilation and transport of nitrogen compounds by plants. Soil Biol. Biochem 5, 109–119 (1973). [Google Scholar]
  22. Daniel-Vedele F., Krapp A. & Kaiser W. M. in Plant Cell Monographs. (eds. Hell R., & Mendel R. R.) 145–172 (2010).
  23. Simard S. W., Durall D. M. & Jones M. D. Carbon allocation and carbon transfer between Betula papyrifera and Pseudotsuga menziesii seedlings using a 13C pulse-labeling method. Plant Soil 191, 41–55 (1997). [Google Scholar]
  24. Moore-Kucera J. & Dick R. P. A pulse-chase method to 13carbon-label Douglas-fir seedlings for decomposition studies. Soil Sci. 173, 46–53 (2008). [Google Scholar]
  25. Bird J. A. & Torn M. S. Fine roots vs. needles: a comparison of 13C and 15N dynamics in a ponderosa pine forest soil. Biogeochemistry 79, 361–382 (2006). [Google Scholar]
  26. Sekiyama Y., Chikayama E. & Kikuchi J. Profiling polar and semipolar plant metabolites throughout extraction processes using a combined solution-state and high-resolution magic angle spinning NMR approach. Anal. Chem. 82, 1643–1652 (2010). [DOI] [PubMed] [Google Scholar]
  27. Earl W. L. & VanderHart D. L. Measurement of 13C chemical shifts in solids. J. Magn. Reson. 48, 35–54 (1982). [Google Scholar]
  28. Hayashi S. & Hayamizu K. Chemical shift standards in high-resolution solid-state NMR (2) 15N Nuclei. Bull. Chem. Soc. Jpn. 64, 688–690 (1991). [Google Scholar]

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