Abundance 13C Research Articles

Deciphering the dual role of bacterial communities in stabilizing rhizosphere priming effect under intra-annual change of growing seasons

The rhizosphere priming effect (RPE) is a widely observed phenomenon affecting carbon (C) turnover in plant-soil systems. While multiple cropping and seasonal changes can have significant impacts on RPE, the mechanisms driving these processes are complex and not yet fully understood. Here, we planted maize in paddy soil during two growing seasons having substantial temperature differences [May–August (warm season, 26.6 °C) and September–November (cool season, 23.1 °C)] within the same calendar year in southern China to examine how seasonal changes affect RPEs and soil C. We identified sources of C emissions by quantifying the natural abundance of 13C and determined microbial metabolic limitations or efficiency and functional genes related to C cycling using an enzyme-based biogeochemical equilibrium model and high-throughput quantitative PCR-based chip technology, respectively. Results showed that microbial metabolism was mainly limited by phosphorus in the warm season, but by C in the cool season, resulting in positive RPEs in both growing seasons, but no significant differences (9.02 vs. 6.27 mg C kg−1 soil day−1). The RPE intensity remained stable as temperature increased (warm season compared to a cool season), which can be largely explained by the simultaneous increase in the abundance of functional genes related to both C degradation and fixation. Our study highlights the simultaneous response and adaptation of microbial communities to seasonal changes and hence contributes to an understanding and prediction of microbially mediated soil C turnover under multiple cropping systems.

Stable C and N isotope abundances in water-extractable organic matter from air-dried soils as potential indices of microbially utilized organic matter

Stable carbon (C) and nitrogen (N) isotopes (13C and 15N) in water-extractable organic matter (WEOM) derived from air-dried soils may be applicable to elucidate the microbial decomposition of soil organic matter (SOM), which is crucial in terrestrial C cycles. A total of 40 soil samples were collected from a depth of 0–6 cm from a temperate broadleaved forest in Japan with vegetation succession from grassland approximately 150 years ago. Those soil samples were air-dried before the water extraction process and organic matter analysis. The C and N concentrations of WEOM were <3.6% of those of the bulk soil and were positively correlated with those of the bulk soil at a p-value of < 0.01. A positive correlation between the two fractions (i.e., WEOM and bulk soils) was also found for natural 13C and 15N abundances (δ13C and δ15N; p < 0.01). However, the C/N ratio of WEOM was slightly correlated with that of bulk soils, exhibiting a narrow range of values of ~10. Thus, those features of the WEOM were similar to the well-known features of microbial biomass. The δ13C and δ15N enrichments in WEOM relative to bulk soil, the difference in stable isotope abundances between bulk SOM and WEOM were negatively and positively correlated, respectively, with the concentrations of organo-mineral complexes and short-range order minerals (non-crystalline oxyhydroxides of aluminum and iron, allophane, imogolite, and allophane-like constituents), which play significant roles in SOM stabilization in soils. These relationships suggest that the stable isotopic enrichments in WEOM can be a good indicator of the microbial utilization of soil C and N under different substrate availabilities, which are crucial to SOM decomposition and decomposability substantially varying from local to global scales.

NMR-Based Method for Intramolecular 13C Distribution at Natural Abundance Adapted to Small Amounts of Glucose.

Quantitative nuclear magnetic resonance (NMR) for isotopic measurements, known as irm-NMR (isotope ratio measured by NMR), is well suited for the quantitation of 13C-isotopomers in position-specific isotope analysis and thus for measuring the carbon isotope composition (δ13C, mUr) in C-atom positions. Irm-NMR has already been used with glucose after derivatization to study sugar metabolism in plants. However, up to now, irm-NMR has exploited a "single-pulse" sequence and requires a relatively large amount of material and long experimental time, precluding many applications with biological tissues or extracts. To reduce the required amount of sample, we investigated the use of 2D-NMR analysis. We adapted and optimized the NMR sequence so as to be able to analyze a small amount (10 mg) of a glucose derivative (diacetonide glucofuranose, DAGF) with a precision better than 1 mUr at each C-atom position. We also set up a method to correct raw data and express 13C abundance on the usual δ13C scale (δ-scale). In fact, due to the distortion associated with polarization transfer and spin manipulation during 2D-NMR analyses, raw 13C abundance is found to be on an unusual scale. This was compensated for by a correction factor obtained via comparative analysis of a reference material (commercial DAGF) using both previous (single-pulse) and new (2D) sequences. Glucose from different biological origins (CO2 assimilation metabolisms of plants, namely, C3, C4, and CAM) was analyzed with the two sequences and compared. Validation criteria such as selectivity, limit of quantification, precision, trueness, and robustness are discussed, including in the framework of green analytical chemistry.

Metabolic labeling of hyaluronan: Biosynthesis and quantitative analysis of 13C,15N-enriched hyaluronan by NMR and MS-based methods

Hyaluronan (HA), a member of the GAG family of glycans, has many diverse biological functions that vary a lot depending on the length of the HA chain and its concentration. A better understanding of the structure of different-sized HA at the atomic level is therefore crucial to decipher these biological functions. NMR is a method of choice for conformational studies of biomolecules, but there are limitations due to the low natural abundance of the NMR active nuclei 13C and 15N. We describe here the metabolic labeling of HA using the bacterium Streptococcus equi subsp. Zooepidemicus and the subsequent analysis by NMR and mass spectrometry. The level of 13C and 15N isotope enrichment at each position was determined quantitatively by NMR spectroscopy and was further confirmed by high-resolution mass spectrometry analysis. This study provides a valid methodological approach that can be applied to the quantitative assessment of isotopically labeled glycans and will help improve detection capabilities and facilitate future structure-function relationship analysis of complex glycans.

Low-temperature at booting reduces starch content and yield of wheat by affecting dry matter transportation and starch synthesis.

With the continuous change of global climate, the frequency of low-temperature stress (LTS) in spring increased greatly, which led to the increase of wheat yield decline. The effects of LTS at booting on grain starch synthesis and yield were examined in two wheat varieties with differing low-temperature sensitivities (insensitive variety Yannong 19 and sensitive variety Wanmai 52). A combination of potted and field planting was employed. For LTS treatment at booting, the wheat plants were placed in a climate chamber for 24h at -2°C, 0°C or 2°C from 19:00 to 07:00 then 5°C from 07:00 to 19:00. They were then returned to the experimental field. The effects of flag leaf photosynthetic characteristics, the accumulation and distribution of photosynthetic products, enzyme activity related to starch synthesis and relative expression, the starch content, and grain yield were determined. LTS at booting caused a significant reduction in the net photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr) of the flag leaves at filling. The development of starch grains in the endosperm is also hindere, there are obvious equatorial grooves observed on the surface of the A-type starch granules, and a reduction in the number of B-type starch granules. The abundance of 13C in the flag leaves and grains decreased significantly. LTS also caused a significant reduction in translocation amount of pre-anthesis stored dry matte from vegetative organs to grains and amount of post-anthesis transfer of accumulated dry matte into grains, and the distribution rate of dry matter in the grains at maturity. The grain filling time was shortened, and the grain filling rate decreased. A decrease in the activity and relative expression of enzymes related to starch synthesis was also observed, with a decrease in the total starch content. As a result, a decrease in the grain number per panicle and 1000-grain weight were also observed. These findings highlight the underlying physiological cause of decreased starch content and grain weight after LTS in wheat.

Maize residue types and soil fertility levels influence sequestration of newly associated carbon in aggregates with in situ experiments

Soil physical structure plays a key role in the sequestration of organic carbon (C) and affects soil fertility and global climate change. However, the dynamics of the residue-derived C in different aggregate fractions are poorly understood. The objectives of this study were to explore the changes in soil aggregate composition and organic C distribution at different fertility levels after the addition of maize residues (roots, stems, and leaves, labeled with 13C). In this study, soil samples with high fertility (HF) and low fertility (LF) levels were formed through a long-term fertilization experiment (29 years), and different types of maize residues were added to the two types of soils. The 13C abundance was quantified in four fractions of soil aggregates large macroaggregates (>2 mm), small macroaggregates (0.25–2 mm), microaggregates (0.053–0.25 mm), and silt and clay (<0.053 mm) on the 60th, 90th, 180th and 540th day following the addition of residues. The findings indicate that the mass proportions of small macroaggregates and microaggregates in the LF soil were lower, whereas those of the other fractions were higher compared with HF soil. Moreover, greater amount of residue-derived C was found to be associated with soil aggregates in LF soil than that in HF soil, and the residue-derived C in different soil aggregate fractions differed with the residue type. The percentages of residue-derived C distributed in the large macroaggregate fractions averaged 19.9% and 15.3% in the LF and HF soils, respectively. Stem and leaf treatments of HF soil improved the stability of soil aggregates. The addition of roots significantly increased the soil organic C content in the aggregates; the residue-derived C content was the highest in the silt and clay fractions and the lowest in the large macroaggregate fraction (>2 mm). These results suggest that the potential benefits of adding maize residue to regulate SOC sequestration and stabilization of soil aggregates, especially in infertile soils.

Borosilicate glass media for neutron sensing

This work using a borosilicate glass medium seeks to detect photoneutrons generated by a medical linear accelerator. The studies involved an Elekta Infiniti accelerator located in the Oncology Centre, Medical City, Riyadh, the neutrons resulting from interactions at above-threshold therapeutic photon energies. The borosilicate detectors were produced from microscope glass slides of nominal thickness 1.0 mm, the latter made into square segments of area 0.6 × 0.6 cm2, a size capable of being accommodated within the planchette of the thermoluminescent (TLD) Reader (Harshaw 600). With some 4% boron in the coverslip glass and the abundance of 10B being 19.8%, thermalised neutrons 10B(n,α)7Li interactions offer a cross-section that can achieve a value as large as 3837 b. Apart from interactions in boron, of additional note is that 2H(γ,n)1H and 9Be(γ,n)8Be are two further photoneutron reactions, giving rise to particularly low threshold energies at 2.226 and 1.666 MeV, respectively. The presence of natural carbon in the present borosilicate samples has been measured to be 1.3%. Accordingly, with the abundance of 13C in natural carbon being 1.109% this would appear to be of very little interest herein. Moreover, compositional measurements made on the borosilicate coverslip glass indicates 9.40 Na. The induced activity in the glass falls to some 25% 30 h post-irradiation, a delay time beyond which readout can subsequently be conducted.

Production of isotopically enriched high molecular weight hyaluronic acid and characterization by solid-state NMR

Hyaluronic acid (HA) is a naturally occurring polysaccharide that is abundant in the extracellular matrix (ECM) of all vertebrate cells. HA-based hydrogels have attracted great interest for biomedical applications due to their high viscoelasticity and biocompatibility. In both ECM and hydrogel applications, high molecular weight (HMW)-HA can absorb a large amount of water to yield matrices with a high level of structural integrity. To understand the molecular underpinnings of structural and functional properties of HA-containing hydrogels, few techniques are available. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for such studies, e.g. 13C NMR measurements can reveal the structural and dynamical features of (HMW) HA. However, a major obstacle to 13C NMR is the low natural abundance of 13C, necessitating the generation of HMW-HA that is enriched with 13C isotopes. Here we present a convenient method to obtain 13C- and 15N-enriched HMW-HA in good yield from Streptococcus equi subsp. zooepidemicus. The labeled HMW-HA has been characterized by solution and magic angle spinning (MAS) solid-state NMR spectroscopy, as well as other methods. These results will open new ways to study the structure and dynamics of HMW-HA-based hydrogels, and interactions of HMW-HA with proteins and other ECM components, using advanced NMR techniques.

Hyperpolarized 13C NMR Spectroscopy of Urine Samples at Natural Abundance by Quantitative Dissolution Dynamic Nuclear Polarization

AbstractHyperpolarized nuclear magnetic resonance (NMR) offers an ensemble of methods that remarkably address the sensitivity issues of conventional NMR. Dissolution Dynamic Nuclear Polarization (d‐DNP) provides a unique and general way to detect 13C NMR signals with a sensitivity enhanced by several orders of magnitude. The expanding application scope of d‐DNP now encompasses the analysis of complex mixtures at natural 13C abundance. However, the application of d‐DNP in this area has been limited to metabolite extracts. Here, we report the first d‐DNP‐enhanced 13C NMR analysis of a biofluid ‐urine‐ at natural abundance, offering unprecedented resolution and sensitivity for this challenging type of sample. We also show that accurate quantitative information on multiple targeted metabolites can be retrieved through a standard addition procedure.

Can N2 fixation by forage legumes build soil organic matter to rival fertilizer N in a tropical forest biome?

Fertilization of pastures with nitrogen (N) can increase animal productivity and may also increase the stocks of soil carbon (C). Recently stoloniferous forage legumes have been used to develop productive sustainable mixed pastures in the Atlantic forest biome. The objective of this study was to compare the impact over 24 years on soil C and N stocks of a productive N-fertilized pasture of Urochloa brizantha cv. Marandu with a mixed forage peanut (Arachis pintoi)-Marandu grass pasture introduced 8 years after an N-fertilized elephantgrass (Pennisetum purpureum) pasture. The pastures were rotationally stocked by Nellore cattle a green-forage allowance equivalent to 4% of body weight and animal performance was continuously monitored. Soil samples were taken at the start of the study and after 8 and 24 years of grazing and analyzed for total C and N and 13C abundance. In 8 years, the stocks of soil organic matter (SOM – 0-30 cm) increased by 4.4 Mg C and 0.58 Mg N ha−1 under the elephantgrass pasture and by 10.4 Mg C and 1.23 Mg N ha−1 under the Marandu grass pasture, both fertilized with 160 kg N ha−1 yr−1. Over the subsequent 16 years, SOM stocks under the Marandu grass fertilized with 120 kg N ha−1 and the mixed forage peanut-Marandu pasture which replaced elephantgrass attained similar stocks of soil C of 51.8 and 52.5 Mg C ha−1 respectively, approximately 12 Mg C greater than under the native vegetation. The 13C abundance of the soil samples indicated that the C4 grass roots made an insignificant contribution to soil C below 30 cm depth. The results show that the N2-fixing forage peanut was just as effective in accumulating SOM as an annual application of 120 kg N fertilizer ha−1 and indicated that after 24 years of these pastures there was little further SOM accumulation.

SEQUENTIAL THERMAL ANALYSIS OF COMPLEX ORGANIC MIXTURES: PROCEDURAL STANDARDS AND IMPROVED CO2 PURIFICATION CAPACITY

ABSTRACTSequential thermal analysis allows for deconvoluting the refractory nature and complexity of carbon mixtures embedded in mineral matrices for subsequent offline stable carbon and radiocarbon (14C) isotope analyses. Originally developed to separate Holocene from more ancient sedimentary organic matter to improve dating of marine sediments, the Ramped Pyrolysis and Oxidation (RPO) apparatus, or informally, the “dirt burner” is now used to address pressing questions in the broad field of biogeochemistry. The growing interest in the community now necessitates improved handling and procedures for routine analyses of difficult sample types. Here we report on advances in CO2 purification during sample processing, modifications to the instrumentation at the National Ocean Sciences Accelerator Mass Spectrometry (NOSAMS) facility, and introduce sodium bicarbonate procedural standards with differing natural abundance 14C signatures for blank assessment. Measurements from different environmental samples are used to compare the procedure to the different generations of sequential thermal analyses. With this study, we aim to improve the standardization of the procedures and prepare this instrumentation for innovations in online stable carbon isotopes and direct AMS-interface measurements in the future.

Natural &lt;sup&gt;13&lt;/sup&gt;C Abundance of Organic Matter in Water-Stable Aggregates of Haplic Chernozem under Conditions of Contrasting Land Uses

Natural 13C abundance of different organic matter (OM) pools in water-stable macro- and free microaggregates of Haplic Chernozem in contrasting land use variants (steppe and long-term bare fallow) are described. Fractionation of 13C at certain stages of the formation of OM pools is relatively constant, regardless of the level of structural organization. This is demonstrated by the presented conceptual scheme, which allows one to quantify the fluxes of carbon (C) in the system of aggregate/OM pool. It was revealed that the main C fluxes in the OM pools go from the free OM (LFfr) to the Residue fraction through microaggregates within water-stable aggregates (mWSA), the components of which are the occluded OM (LFocc) and the Clay. With a high degree of probability, C flows from macroaggregates (WSAma) to free microaggregates (WSAmi). At the same time, the higher probability of the C flux into the Residue from the mWSA as compared to the direct C fluxes from the LFocc and the Clay, testifies in favor of the hypothesis that the Residue is represented, to a large extent, by parts/fragments of disintegrated mWSAs of 50–1 μm in size. Regardless of the size, the WSA contains a labile OM (as the components of mWSA, along with LFfr – only in macroaggregates) and a stable OM (Residue). Labile OM (LFocc and Clay) within WSAmi is characterized by a lower degree of microbial processing (“lighter” isotopic signature) compared to that within WSAma, which is due to its greater “physical” protection against microbial attacks. However, the most stable OM pool, concentrated in the Residue within WSAmi is enriched in 13C compared to that within WSAma. Considering that the Residue determines the total isotopic composition of C in WSA, the organic matter of free microaggregates is characterized by a higher degree of microbial processing in comparison with that of macroaggregates. Free microaggregates are parts of disintegrated macroaggregates.

Natural 13C Abundance in the Organic Matter of Water-Stable Aggregates of Haplic Chernozem under Contrasting Land Uses

Natural 13C Abundance in the Organic Matter of Water-Stable Aggregates of Haplic Chernozem under Contrasting Land Uses

Effect of light conditions on trophic level and gene expression of partially mycoheterotrophic orchid, Cymbidium goeringii

ABSTRACT Partial mycoheterotrophic i.e., mixotrophic, plants are the species which partially depend on mycorrhizal fungi for its nutrients. Although some of these plants are known to show plasticity in the degree of fungal dependence induced by the changes in light condition, the genetic background of this plasticity is largely unsolved. Here, we investigated the relationships between environmental conditions and nutrient sources based on 13C and 15N enrichment in mixotrophic orchid Cymbidium goeringii. We also shaded them for 2 months and evaluated the effect of light condition on the nutrient sources based on the abundance of 13C and 15N and the gene expressions by RNA-seq based de novo assembly. The shading had no effect on isotope enrichment, possibly because of the translocation of carbon and nitrogen from the storage organs. Gene expression analysis showed the upregulation of genes involved in jasmonic acid response in leaves of the shaded plants, which suggests that the jasmonic acid played an important role in regulation of degree of dependence against the mycorrhizal fungi. Our results suggest that mixotrophic plants might be controlling their dependency against the mycorrhizal fungi by a common mechanism with the autotrophic plants.

One Ca2+ Site in CaNaY Zeolite Can Attach Three CO2 Molecules.

To design efficient CO2 capture materials, it is necessary to ensure a high adsorption capacity. We recently reported that one Na+ site in NaY zeolite can attach two CO2 molecules. However, the process is not suitable for practical use because it proceeds at a low temperature. Here, we present results on CO2 adsorption on CaNaY zeolites, demonstrating that one Ca2+ site can attach three CO2 molecules. The ν3(13CO2) mode arising from the natural 13C abundance allows for easy infrared monitoring of the processes: it appears at 2298, 2294, and 2291 cm-1 for the complexes with one, two, and three CO2 ligands, respectively. The 12CO2 molecules in the polyligand complexes interact vibrationally, leading to the split of the ν3(12CO2) modes. At ambient temperature, Ca2+(CO2)2 complexes predominate at >1 mbar CO2 and triligand species begin to form at 65 mbar. The obtained results show that CaY zeolites can be very effective CO2 capture materials.

Comparison of Total Body Vitamin A Stores Using Individual versus Population 13C-Natural Abundance of Serum Retinol in Preschool Children and Women Residing in 6 Diverse African Countries

BackgroundStable isotope techniques using 13C to assess vitamin A (VA) dietary sources, absorption, and total body VA stores (TBSs) require determination of baseline 13C abundance. 13C-natural abundance is approximately 1.1% total carbon, but varies with foods consumed, supplements taken, and food fortification with synthetic retinyl palmitate. ObjectivesWe determined 13C variation from purified serum retinol and the resulting impact on TBSs using pooled data from preschool children in Burkina Faso, Cameroon, Ethiopia, South Africa, Tanzania, and Zambia and Zambian women. MethodsSeven studies included children (n = 639; 56 ± 25 mo; 48% female) and one in women (n = 138; 29 ± 8.5 y). Serum retinol 13C-natural abundance was determined using GC-C-IRMS. TBSs were available in 7 studies that employed retinol isotope dilution (RID). Serum CRP and α1-acid-glycoprotein (AGP) were available from 6 studies in children. Multivariate mixed models assessed the impact of covariates on retinol 13C. Spearman correlations and Bland–Altman analysis compared serum and milk retinol 13C and evaluated the impact of using study- or global-retinol 13C estimates on calculated TBSs. Results13C-natural abundance (%, median [Q1, Q3]) differed among countries (low: Zambia, 1.0744 [1.0736, 1.0753]; high: South Africa, 1.0773 [1.0769, 1.0779]) and was associated with TBSs, CRP, and AGP in children and with TBSs in women. 13C-enrichment from serum and milk retinol were correlated (r = 0.52; P = 0.0001). RID in children and women using study and global estimates had low mean bias (range, −3.7% to 2.2%), but larger 95% limits of agreement (range, −23% to 37%). Conclusions13C-natural abundance is different among human cohorts in Africa. Collecting this information in subgroups is recommended for surveys using RID. When TBSs are needed on individuals in clinical applications, baseline 13C measures are important and should be measured in all enrolled subjects.

Quantification and isotope abundance determination of 13C labeled intracellular sugar metabolites with hydrophilic interaction liquid chromatography.

Metabolic flux analysis (MFA) using stable isotope labeled tracers is a powerful tool to estimate fluxes through metabolic pathways. It finds applications in studying metabolic changes in diseases, regulation of cellular energetics, and novel strategies for metabolic engineering. Accurate and precise quantification of the concentration of metabolites and their labeling states is critical for correct MFA results. Utilizing an ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) system, an analytical method for simultaneously quantifying the concentration of sugar metabolites and their mass isotopologue distribution (MID) was developed. The method performs with good linearity and coefficient of determination (R2) > 0.99, while the detection limit ranged from 0.1 to 50 mg L-1. Seven sugar metabolites were detected in a labeled Brevibacterium flavum sample using the method. The detected quantities ranged from 6.15 to 3704.21 mg L-1, and 13C abundance was between 12.77% and 66.67% in the fermentation fluid and 16.28% and 91.93% in the bacterial body. Overall, the method is efficient, accurate, and suitable for analysis of labeled sugar metabolites in 13C MFA studies.

Amorphous iron oxides protect aggregate-associated organic carbon from microbial utilization and decomposition evidenced from the natural abundance of 13C

Iron oxides play a critical role in promoting organic carbon sequestration through adsorption or co-precipitation. However, the effects of iron oxide types on the stabilization of aggregate-associated organic carbon are still poorly understood. We investigated the distribution of crystalline iron oxides (Fec), amorphous iron oxides (Feo), δ13C and Fe-bound OC in soil aggregate fractions of red earth (Ferrisol). The results showed that the δ13C values in microaggregate and silt+clay fractions were significantly lower than macro- and mesoaggregate (p < 0.05). The contents of Feo and Fe-bound OC in the microaggregate and silt+clay fractions were significantly higher than macro- and masoaggregate fractions (p < 0.05), while the content of Fec increased with the increase of aggregate size. The OC: Fe molar ratio of Feo was higher than Fec in all aggregate fractions. The FTIR spectroscopy analyses showed that polysaccharides had the highest proportion of all OC functional groups and increased with the increase of aggregate size. Likewise, the ratio of aromatic-C/aliphatic-C increased with the increase of aggregate size. Both Feo and Fe-bound OC had a significant negative correlation with δ13C values in microaggregate and silt+clay fractions, while Fec had no significant correlation with δ13C values in all aggregate fractions. Furthermore, the Fe-bound OC and fFe-bound OC were positively correlated with Feo and negatively correlated with Fec in all aggregate fractions. This study highlighted the importance of Feo in the sequestration of aggregate-associated organic carbon and confirmed the hypothesis that the SOC in microaggregates and silt+clay underwent less cycles of microbial utilization due to the protection of Feo, thereby limiting the molecular structure complexity and aromaticity of SOM.

Natural 13C Abundance of Organic-Matter Pools in Haplic Chernozem

Natural 13C Abundance of Organic-Matter Pools in Haplic Chernozem

Elucidation of lignin and polysaccharide linkages in wheat straw by 2H/13C isotopic tracer

To elucidate chemical linkages between lignin and polysaccharides, the aqueous mixed solutions of coniferin-[α-13C], syringin-[α-13C], D-glucose-[6-2H], and phenylalanine ammonia-lyase inhibitor were injected into a living wheat stalk. Internode tissues with high abundance of 2H-13C were collected. The milled wood lignin, lignin-carbohydrate complex (LCC), and residual LCC (R-LCC) with enrichment of 2H-13C were isolated. The 13C and 2H abundances showed that the lignin and polysaccharides of internode tissues were labeled by 13C and 2H, respectively. Analysis with carbon-13 nuclear magnetic resonance (13C-NMR) showed that ketal and benzyl ether bonds were formed between α-C of lignin and carbohydrates. The R-LCC and LCC were further treated with enzymes to obtain enzymatic degraded R-LCC (ED-R-LCC) and enzymatic degraded LCC (ED-LCC). 13C-NMR spectra of ED-LCC showed that the α-C of lignin side chain was combined with 6-C of carbohydrates by ether, ester, and ketal linkages. 1H-NMR differential spectra of ED-LCCs revealed an LC linkage of benzyl ether bond. Glucan-lignin (En-R-GL) and xylan-lignin (En-R-XL) complexes were separated from ED-R-LCC by ionic liquid. A part of lignin α-C was linked to cellulose 6-C by benzyl ether and α-ketal linkages. 13C-NMR spectra of En-R-XL showed there were α-benzyl ether and α-ketal bonds between lignin and xylan.