Dual,differential isotope labeling shows the preferential movement of labile plant constituents into mineral‐bonded soil organic matter

The formation and stabilization of soil organic matter (SOM) are major concerns in the context of global change for carbon sequestration and soil health. It is presently believed that lignin is not selectively preserved in soil and that chemically labile compounds bonding to minerals comprise a large fraction of the SOM. Labile plant inputs have been suggested to be the main precursor of the mineral‐bonded SOM. Litter decomposition and SOM formation are expected to have temperature sensitivity varying with the lability of plant inputs. We tested this framework using dual ¹³C and ¹⁵N differentially labeled plant material to distinguish the metabolic and structural components within a single plant material. Big Bluestem (Andropogon gerardii) seedlings were grown in an enriched ¹³C and ¹⁵N environment and then prior to harvest, removed from the enriched environment and allowed to incorporate natural abundance ¹³C–CO₂ and ¹⁵N fertilizer into the metabolic plant components. This enabled us to achieve a greater than one atom % difference in ¹³C between the metabolic and structural components within the plant litter. This differentially labeled litter was incubated in soil at 15 and 35 °C, for 386 days with CO₂ measured throughout the incubation. After 14, 28, 147, and 386 days of incubation, the soil was subsequently fractionated. There was no difference in temperature sensitivity of the metabolic and structural components with regard to how much was respired or in the amount of litter biomass stabilized. Only the metabolic litter component was found in the sand, silt, or clay fraction while the structural component was exclusively found in the light fraction. These results support the stabilization framework that labile plant components are the main precursor of mineral‐associated organic matter.     

Relative and accurate measurement of protein abundance using 15N stable isotope labeling in Arabidopsis (SILIA)

In the quantitative proteomic studies, numerous in vitro and in vivo peptide labeling strategies have been successfully applied to measure differentially regulated protein and peptide abundance. These approaches have been proven to be versatile and repeatable in biological discoveries. ¹⁵N metabolic labeling is one of these widely adopted and economical methods. However, due to the differential incorporation rates of ¹⁵N or ¹⁴N, the labeling results produce imperfectly matched isotopic envelopes between the heavy and light nitrogen-labeled peptides. In the present study, we have modified the solid Arabidopsis growth medium to standardize the ¹⁵N supply, which led to a uniform incorporation of ¹⁵N into the whole plant protein complement. The incorporation rate (97.43 ± 0.11%) of ¹⁵N into ¹⁵N-coded peptides was determined by correlating the intensities of peptide ions with the labeling efficiencies according to Gaussian distribution. The resulting actual incorporation rate (97.44%) and natural abundance of ¹⁵N/¹⁴N-coded peptides are used to re-calculate the intensities of isotopic envelopes of differentially labeled peptides, respectively. A modified ¹⁵N/¹⁴N stable isotope labeling strategy, SILIA, is assessed and the results demonstrate that this approach is able to differentiate the fold change in protein abundance down to 10%. The machine dynamic range limitation and purification step will make the precursor ion ratio deriving from the actual ratio fold change. It is suggested that the differentially mixed ¹⁵N-coded and ¹⁴N-coded plant protein samples that are used to establish the protein abundance standard curve should be prepared following a similar protein isolation protocol used to isolate the proteins to be quantitated.     

Dictyostelium discoideum as Expression Host: Isotopic Labeling of a Recombinant Glycoprotein for NMR Studies

The advantages of the organism Dictyostelium discoideum as an expression host for recombinant glycoproteins have been exploited for the production of an isotopically labeled cell surface protein for NMR structure studies. Growth medium containing [¹⁵N]NH₄Cl and [¹³C]glycerol was used to generate isotopically labeled Escherichia coli, which was subsequently introduced to D. discoideum cells in simple Mes buffer. A variety of growth conditions were screened to establish minimal amounts of nitrogen and carbon metabolites for a cost-effective protocol. Following single-step purification by anion-exchange chromatography, 8 mg of uniformly ¹³C,¹⁵N-labeled protein secreted by approximately 10¹⁰D. discoideum cells was isolated from 3.3 liters of supernatant. Mass spectrometry showed the recombinant protein of 16 kDa to have incorporated greater than 99.9% isotopic label. The two-dimensional ¹H-¹³C HSQC spectrum confirms ¹³C labeling of both glycan and amino acid residues of the glycoprotein. All heteronuclear NMR spectra showed a good dispersion of cross-peaks essential for high-quality structure determination.     

Amino acids as a nitrogen source for tomato seedlings: The use of dual-labeled (C, N) glycine to test for direct uptake by tomato seedlings

Direct uptake of organic nitrogen (ON) compounds, rather than inorganic N, by plant roots has been hypothesized to constitute a significant pathway for plant nutrition. The aim of this study was to test whether tomatoes (Solanum lycopersicum cv. Huying932) can take up ON directly from the soil by using ¹⁵NH₄Cl, K¹⁵NO₃, 1, 2-¹³C₂¹⁵N-glycine labeling techniques. The ¹³C and ¹⁵N in the plants increased significantly indicating that a portion of the glycine-N was taken up in the form of intact amino acids by the tomatoes within 48 h after injection into the soil. Regression analysis of excess ¹³C against excess ¹⁵N showed that approximately 21% of the supplied glycine-N was taken up intact by the tomatoes. Atom% excesses of ¹⁵N and ¹³C in the roots were higher than in any shoots. Results also indicated rapid turnover of amino acids (e.g., glycine) by soil microorganisms, and the poor competitive ability of tomatoes in absorbing amino acids from the soil solution. This implies that tomatoes can take up ON in an intact form from the soil despite the rapid turnover of organic N usually found under such conditions. Given the influence of climatic change and N pollution, further studies investigating the functional ecological implications of ON in horticultural ecosystems are warranted.     

A method for efficient isotopic labeling of recombinant proteins

A rapid and efficient approach for preparing isotopically labeled recombinant proteins is presented. The method is demonstrated for ¹³C labeling of the C-terminal domain of angiopoietin-2, ¹⁵N labeling of ubiquitin and for ²H/¹³C/¹⁵N labeling of the Escherichia coli outer-membrane lipoprotein Lpp-56. The production method generates cell mass using unlabeled rich media followed by exchange into a small volume of labeled media at high cell density. Following a short period for growth recovery and unlabeled metabolite clearance, the cells are induced. The expression yields obtained provide a fourfold to eightfold reduction in isotope costs using simple shake flask growths.     

Patterns of soil and plant nitrogen isotope composition and their relationships with climate factors in Xilingol League,Inner Mongolia,China

Background: Plant and soil nitrogen stable isotope (δ¹⁵N) can integrate several fundamental biogeochemical processes in ecosystem nitrogen dynamics, and reflect characteristics of ecosystem nitrogen cycling.

Aims: We investigated how climate change influenced plant-soil nitrogen cycling by relating soil δ¹⁵N, plant δ¹⁵N and Δδ¹⁵N (difference between soil and plant δ¹⁵N) with climatic factors.

Methods: Field investigation was conducted in temperate grasslands in Inner Mongolia during August 2015. Plant δ¹⁵N, soil δ¹⁵N and Δδ¹⁵N were determined, and their relationships with climatic factors were examined by simple regression analyses and general linear models.

Results: Soil δ¹⁵N was significantly higher than plant δ¹⁵N, and there was a positive linear correlation between them. Soil and plant δ¹⁵N were negatively related with mean annual precipitation (MAP) and positively with mean annual temperature (MAT); conversely, Δδ¹⁵N was positively related with MAP and negatively with MAT.

Conclusion: Soil δ¹⁵N was dominantly controlled by MAT, while it was MAP for plant δ¹⁵N. Climate factors influenced plant δ¹⁵N not only through their effects on soil nitrogen dynamics but also strategies of plant nitrogen acquisition. Thus, compared with plant δ¹⁵N, soil δ¹⁵N can more accurately reflect soil nitrogen dynamics, while plant δ¹⁵N may integrate soil nitrogen dynamics and plant nitrogen acquisition.     

Variation in the export of 13C and 15N from soybean leaf: the effects of nitrogen application and sink removal

Translocation of carbon and nitrogen within a single source-sink unit, comprising a trifoliated leaf, the axillary pod and the subtending internode, and from this unit to the rest of the plant was examined in soybean (Glycine max L. cv. Akishirome) plant by feeding ¹³CO₂ and ¹⁵NO₃. The plants were grown at two levels of nitrogen in the basal medium, i.e. low-N (2 g N m^–2) and high-N (35 g N m^–2) and a treatment of depodding was imposed by removing all the pods from the plant, except the pod of the source sink unit, 13 days after flowering. The plants at high-N accumulated more biomass in its organs compared to low-N and pod removal increased the weight of the vegetative organs. When the terminal leaflet of the source-sink unit was fed with ¹³CO₂, almost all of the radioactive materials were retained inside the source-sink unit and translocation to rest of the plants was insignificant under any of the treatments imposed. Out of the¹³C exported by the terminal leaflet, less than half went into the axillary pod, as the lateral leaflets claimed equal share and very little material was deposited in the petiole. Pod removal decreased ¹³C export at high-N , but not at low-N. Similar to ¹³C, the source-sink unit retained all the ¹⁵N fed to the terminal leaflet at high-N. At low-N, the major part of ¹⁵N partitioning occurred in favour of the rest of the plant outside the source-sink unit, but removal of the competitve sinks from the rest of the plants nullified any partitioning outside the unit. Unlike the situation in ¹³C, no partitioning of ¹⁵N occurred in favour of the lateral leaflets from the terminal leaflet inside the unit. It is concluded that sink demand influences partitioning of both C and N and the translocation of carbon is different from that of nitrogen within a source-sink unit. The translocation of the N is more adjustive to a demand from other sink units compared to the C.     

Are carbon and nitrogen exchange between fungi and the orchid Goodyera repens affected by irradiance?

Background and Aims The green orchid Goodyera repens has been shown to transfer carbon to its mycorrhizal partner, and this flux may therefore be affected by light availability. This study aimed to test whether the C and N exchange between plant and fungus is dependent on light availability, and in addition addressed the question of whether flowering and/or fruiting individuals of G. repens compensate for changes in leaf chlorophyll concentration with changes in C and N flows from fungus to plant.Methods The natural abundances of stable isotopes of plant C and N were used to infer changes in fluxes between orchid and fungus across natural gradients of irradiance at five sites. Mycorrhizal fungi in the roots of G. repens were identified by molecular analyses. Chlorophyll concentrations in the leaves of the orchid and of reference plants were measured directly in the field.Key Results Leaf δ¹³C values of G. repens responded to changes in light availability in a similar manner to autotrophic reference plants, and different mycorrhizal fungal associations also did not affect the isotope abundance patterns of the orchid. Flowering/fruiting individuals had lower leaf total N and chlorophyll concentrations, which is most probably explained by N investments to form flowers, seeds and shoot.Conclusions The results indicate that mycorrhizal physiology is relatively fixed in G. repens, and changes in the amount and direction of C flow between plant and fungus were not observed to depend on light availability. The orchid may instead react to low-light sites through increased clonal growth. The orchid does not compensate for low leaf total N and chlorophyll concentrations by using a ¹³C- and ¹⁵N-enriched fungal source.     

Arginine bi-directional translocation and breakdown into ornithine along the arbuscular mycorrhizal mycelium

Bi-directional translocation and degradation of Arginine (Arg) along the arbuscular mycorrhizal (AM) fungal mycelium were testified through ¹⁵N and/or ¹³C isotopic labeling. In vitro mycorrhizas of Glomus intraradices and Ri T-DNA-transformed carrot roots were grown in dual compartment Petri dishes. [¹⁵N- and/or¹³C]Arg was supplied to either the fungal compartment or the mycorrhizal compartment or separate dishes containing the uncolonized roots. The levels and labeling of free amino acids (AAs) in the mycorrhizal roots and in the extraradical mycelia(ERM) were measured by gas chromatography/mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). The ERM of AM fungi exposed in either NH₄ ⁺ or urea as sole external nitrogen source had much higher ¹⁵N enrichment of Arg, compared with those in nitrate or exogenous Arg; however, glycerol supplied as an external carbon source to the ERM had no significant effect on the level of Arg in the ERM. Meanwhile, Arg biosynthesized in the ERM could be translocated intact to the mycorrhizal roots and thereby the level of Arg in the mycorrhizal roots increased to about 20% after culture of ERM in 4 mmol/L NH₄ ⁺ for 6 weeks. Also Arg was found to be bi-directionally transported along the AM fungal mycelium through [U-¹³C]Arg labeling either in the mycorrhizal compartment or in the fungal compartment. Once Arg was translocated to the potential N-limited sites, it would be further degraded into ornithine (Orn) and urea since either [U-¹³C] or [U-¹⁵N/U-¹³C]Orn was apparently shown up in the mycorrhizal root tissues when [U-¹³C] or [U-¹⁵N/U-¹³C]Arg was labeled in the fungal compartment, respectively. Evidently Orn formation indicated the ongoing activities of Arg translocation and degradation through the urea cycle in AM fungal mycelium. Supported by Science and Technology Department of Zhejiang Province (Grant No. 2006C22009).     

Fractional <Superscript>13</Superscript>C enrichment of isolated carbons using [1-<Superscript>13</Superscript>C]- or [2-<Superscript>13</Superscript>C]-glucose facilitates the accurate measurement of dynamics at backbone C<Superscript>α</Superscript> and side-chain methyl positions in proteins

A simple labeling approach is presented based on protein expression in [1-¹³C]- or [2-¹³C]-glucose containing media that produces molecules enriched at methyl carbon positions or backbone C^α sites, respectively. All of the methyl groups, with the exception of Thr and Ile(δ1) are produced with isolated ¹³C spins (i.e., no ¹³C–¹³C one bond couplings), facilitating studies of dynamics through the use of spin-spin relaxation experiments without artifacts introduced by evolution due to large homonuclear scalar couplings. Carbon-α sites are labeled without concomitant labeling at C^β positions for 17 of the common 20 amino acids and there are no cases for which ¹³C^α−¹³CO spin pairs are observed. A large number of probes are thus available for the study of protein dynamics with the results obtained complimenting those from more traditional backbone ¹⁵N studies. The utility of the labeling is established by recording ¹³C R _1ρ and CPMG-based experiments on a number of different protein systems.     

Conformational isomerism of the binuclear N,N-pentamethylenedithiocarbamate cadmium(II) complex, [Cd2{S2CN(CH2)5}4] on multinuclear (N, Cd) CP/MAS NMR and single-crystal X-ray diffraction data

Crystalline N,N-cyclo-pentamethylenedithiocarbamate (PmDtc) cadmium(II) complex was prepared and studied by means of ¹⁵N, ¹¹³Cd CP/MAS NMR spectroscopy and single-crystal X-ray diffraction. The unit cell of the cadmium(II) compound comprises two centrosymmetric isomeric binuclear molecules [Cd₂{S₂CN(CH₂)₅}₄], which display structural inequivalence in both ¹⁵N and ¹¹³Cd NMR and XRD data. There are pairs of the dithiocarbamate ligands exhibiting different structural functions in both isomeric molecules. Each of the terminal ligands is bidentately coordinated to the cadmium atom and forms a planar four-membered chelate ring [CdS₂C]; whereas pairs of the tridentate bridging ligands combine two neighbouring cadmium atoms forming an extended eight-membered tricyclic moieties [Cd₂S₄C₂], whose geometry can be approximated by a ‘chair’ conformation. The structural states of cadmium atoms were characterised by almost axially symmetric ¹¹³Cd chemical shift tensors. All experimental ¹⁵N resonance lines were assigned to the nitrogen structural sites in both isomeric binuclear molecules.     

Site-dependent N uptake from N-form mixtures by arctic plants,soil microbes and ectomycorrhizal fungi

Soil microbes constitute an important control on nitrogen (N) turnover and retention in arctic ecosystems where N availability is the main constraint on primary production. Ectomycorrhizal (ECM) symbioses may facilitate plant competition for the specific N pools available in various arctic ecosystems. We report here our study on the N uptake patterns of coexisting plants and microbes at two tundra sites with contrasting dominance of the circumpolar ECM shrub Betula nana. We added equimolar mixtures of glycine-N, NH₄⁺–N and NO₃⁻–N, with one N form labelled with ¹⁵N at a time, and in the case of glycine, also labelled with ¹³C, either directly to the soil or to ECM fungal ingrowth bags. After 2 days, the vegetation contained 5.6, 7.7 and 9.1% (heath tundra) and 7.1, 14.3 and 12.5% (shrub tundra) of the glycine-, NH₄⁺- and NO₃⁻–¹⁵N, respectively, recovered in the plant–soil system, and the major part of ¹⁵N in the soil was immobilized by microbes (chloroform fumigation-extraction). In the subsequent 24 days, microbial N turnover transferred about half of the immobilized ¹⁵N to the non-extractable soil organic N pool, demonstrating that soil microbes played a major role in N turnover and retention in both tundra types. The ECM mycelial communities at the two tundras differed in N-form preferences, with a higher contribution of glycine to total N uptake at the heath tundra; however, the ECM mycelial communities at both sites strongly discriminated against NO₃⁻. Betula nana did not directly reflect ECM mycelial N uptake, and we conclude that N uptake by ECM plants is modulated by the N uptake patterns of both fungal and plant components of the symbiosis and by competitive interactions in the soil. Our field study furthermore showed that intact free amino acids are potentially important N sources for arctic ECM fungi and plants as well as for soil microorganisms. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ammonium Transformation in a Nitrogen-Rich Tidal Freshwater Marsh

The fate and transport of watershed-derived ammonium in a tidal freshwater marsh fringing the nutrient rich Scheldt River, Belgium, was quantified in a whole ecosystem ¹⁵N labeling experiment. In late summer (September) we added ¹⁵N-NH₄⁺ to the flood water entering a 3477 m² tidal freshwater marsh area, and traced the ammonium processing and retention in four subsequent tide cycles. In this paper we present the results for the water-phase components of the marsh system and compare them to a similar experiment conducted in spring/early summer (May). Changes in concentration and isotopic enrichment of NO₃⁻ + NO₂⁻, N₂O, N₂, NH₄⁺ and suspended particulate nitrogen (SPN) were measured in concert with a mass balance study. All analyzed N-pools were labeled, and 49% of the added ¹⁵NH₄⁺ was retained or transformed. The most important pool for ¹⁵N was nitrate, accounting for 17% of ¹⁵N-transformation. N₂, N₂O and SPN accounted for 2.4, 0.02 and 1.4%, respectively. The temporal and spatial patterns of ¹⁵N transformation in the water phase component of the system were remarkably similar to those observed in May, indicating good reproducibility of the whole ecosystem labeling approach, but the absolute ammonium transformation rate was 3 times higher in May. While the marsh surface area was crucial for nitrification in May this was less pronounced in September. Denitrification, on the other hand, appeared more important in September compared to May.     

Specific 15N, NH correlations for residues in15 N, 13C and fractionally deuterated proteins that immediately follow methyl-containing amino acids

A triple-resonance pulse scheme is described which records¹⁵N, NH correlations of residues that immediately follow amethyl-containing amino acid. The experiment makes use of a¹⁵N, ¹³C and fractionally deuterated proteinsample and selects for CH₂D methyl types. The experiment isthus useful in the early stages of the sequential assignment process as wellas for the confirmation of backbone ¹⁵N, NH chemical shiftassignments at later stages of data analysis. A simple modification of thesequence also allows the measurement of methyl side-chain dynamics. This isparticularly useful for studying side-chain dynamic properties in partiallyunfolded and unfolded proteins where the resolution of aliphatic carbon andproton chemical shifts is limited compared to that of amide nitrogens.     

13C labeling of plant assimilates using a simple canopy-scale open air system

A simple system based on web-FACE technology was designed and implemented as an approach to label plant-assimilated carbon (C) with ¹³C. The labeling system avoids the use of a chamber or other enclosure, instead distributing CO₂ heavily enriched in ¹³C at near atmospheric concentrations to the tree foliage through the use of porous tubing. The system was applied to three plantation grown juvenile larch (Larix spp.) trees during the daylight hours over the course of five days in the middle of the growing season. Relative to control trees, fumigation with enriched CO₂ resulted in significantly ¹³C-enriched foliar respiration and nighttime soil respiration. Enrichment was also created in the foliar tissue, but differences between labeled and control trees were not statistically significant. Temporal and spatial variation in the strength of the isotopic label did occur, and modifications to the system are suggested to limit the variation. The approach should enable the implementation of pulse-chase experiments designed to understand plant source-sink relationships or experiments designed to understand the flux of C from plant roots into the soil food web. Responsible Editor: Angela Hodge.     

Nitrogen Transfer Within and Between Plants Through Common Mycorrhizal Networks (CMNs)

Mycorrhizae play a critical role in nutrient capture from soils. Arbuscular mycorrhizae (AM) and ectomycorrhizae (EM) are the most important mycorrhizae in agricultural and natural ecosystems. AM and EM fungi use inorganic NH₄ ⁺ and NO₃ ^?, and most EM fungi are capable of using organic nitrogen. The heavier stable isotope ¹⁵N is discriminated against during biogeochemical and biochemical processes. Differences in ¹⁵N (atom%) or δ¹⁵N (‰) provide nitrogen movement information in an experimental system. A range of 20 to 50% of one-way N-transfer has been observed from legumes to nonlegumes. Mycorrhizal fungal mycelia can extend from one plant's roots to another plant's roots to form common mycorrhizal networks (CMNs). Individual species, genera, even families of plants can be interconnected by CMNs. They are capable of facilitating nutrient uptake and flux. Nutrients such as carbon, nitrogen and phosphorus and other elements may then move via either AM or EM networks from plant to plant. Both ¹⁵N labeling and ¹⁵N natural abundance techniques have been employed to trace N movement between plants interconnected by AM or EM networks. Fine mesh (25～45 μm) has been used to separate root systems and allow only hyphal penetration and linkages but no root contact between plants. In many studies, nitrogen from N₂-fixing mycorrhizal plants transferred to non-N₂–fixing mycorrhizal plants (one-way N-transfer). In a few studies, N is also transferred from non-N₂–fixing mycorrhizal plants to N₂-fixing mycorrhizal plants (two-way N-transfer). There is controversy about whether N-transfer is direct through CMNs, or indirect through the soil. The lack of convincing data underlines the need for creative, careful experimental manipulations. Nitrogen is crucial to productivity in most terrestrial ecosystems, and there are potential benefits of management in soil-plant systems to enhance N-transfer. Thus, two-way N-transfer warrants further investigation with many species and under field conditions.     

Estimation of symbiotically fixed nitrogen in field grown soybeans: An application of natural15N/14N abundance and a low level15N-tracer technique

Summary Plants from agricultural and natural upland ecosystem were investigated for¹⁵N content to evaluate the role of symbiotic N₂-fixation in the nitrogen nutrition of soybean. Increased yields and lower δ¹⁵N values of nodulating soybeansvs, non-nodulating isolines gave semi-quantitative estimates of N₂ fixation. A fairly large discrepancy was found between estimations by δ¹⁵N and by N yield at 0 kg N/ha of fertilizer. More precise estimates were made by following changes in plant δ¹⁵N when fertilizer δ¹⁵N was varied near¹⁵N natural abundance level. Clearcut linear relationships between δ¹⁵N values of whole plants and of fertilizer were obtained at 30 kg N/ha of fertilizer for three kinds of soils. In experimental field plots, nodulating soybeans obtained 13±1% of their nitrogen from fertilizer, 66±8% from N₂ fixation and 21±10% from soil nitrogen in Andosol brown soil; 30%, 16% and 54% in Andosol black soil; 7%, 77% and 16% in Alluvial soil, respectively. These values for N₂ fixation coincided with each corresponding estimation by N yield method. Other results include: 1)¹⁵N content in upland soils and plants was variable, and may reflect differences in the mode of mineralization of soil organics, and 2) nitrogen isotopic discrimination during fertilizer uptake (δ¹⁵N of plant minus fertilizer) ranged from −2.2 to +4.9‰ at 0–30 kg N/ha of fertilizer, depending on soil type and plant species. The proposed method can accurately and relatively simply establish the importance of symbiotic nitrogen fixation for soybeans growing in agricultural settings.     

18O Labeling of Chlorophyll d in Acaryochloris marina Reveals That Chlorophyll a and Molecular Oxygen Are Precursors

The cyanobacterium Acaryochloris marina was cultured in the presence of either H₂¹⁸O or ¹⁸O₂, and the newly synthesized chlorophylls (Chl a and Chl d) were isolated using high performance liquid chromatography and analyzed by mass spectroscopy. In the presence of H₂¹⁸O, newly synthesized Chl a and d, both incorporated up to four isotopic ¹⁸O atoms. Time course H₂¹⁸O labeling experiments showed incorporation of isotopic ¹⁸O atoms originating from H₂¹⁸O into Chl a, with over 90% of Chl a ¹⁸O-labeled at 48 h. The incorporation of isotopic ¹⁸O atoms into Chl d upon incubation in H₂¹⁸O was slower compared with Chl a with ∼50% ¹⁸O-labeled Chl d at 115 h. The rapid turnover of newly synthesized Chl a suggested that Chl a is the direct biosynthetic precursor of Chl d. In the presence of ¹⁸O₂ gas, one isotopic ¹⁸O atom was incorporated into Chl a with approximately the same kinetic incorporation rate observed in the H₂¹⁸O labeling experiment, reaching over 90% labeling intensity at 48 h. The incorporation of two isotopic ¹⁸O atoms derived from molecular oxygen (¹⁸O₂) was observed in the extracted Chl d, and the percentage of double isotopic ¹⁸O-labeled Chl d increased in parallel with the decrease of non-isotopic-labeled Chl d. This clearly indicated that the oxygen atom in the C3¹-formyl group of Chl d is derived from dioxygen via an oxygenase-type reaction mechanism.     

13CO2示踪不同化学形态氮素添加对高寒草甸植物光合碳分配的影响

外源氮素(N)输入陆地生态系统后会引起植物和土壤各碳库的变化,但是对不同化学形态氮素的长期输入如何影响光合碳在植物组织、土壤、土壤呼吸中的分配及转运知之甚少,尤其是对于氮输入引起光合碳分配变化进而作用于植物和土壤碳库的机制的认识还非常匮乏。基于在青藏高原矮嵩草草甸开展的不同化学形态氮素添加的长期实验,利用~(13)C示踪方法揭示了光合碳在植物地上、地下组织的分配,及其随时间在土壤中的滞留和随土壤呼吸的释放。研究结果表明,外源氮素添加10年后,与对照未添加氮素处理相比,氨态氮处理下的地上生物量增加了49.5%,氨态氮处理下的地下生物量增加了111.3%。土壤中滞留的~(13)C整体呈下降趋势,氨态氮处理下的土壤碳库显著高于硝态氮处理下的值。不同处理下的土壤呼吸中~(13)C的滞留量随时间呈指数衰减的变化趋势,其中,硝态氮处理下的~(13)C衰减最快。~(13)C同位素标记后第1天测定植物茎和叶内的~(13)C约占刚刚标定完茎和叶内~(13)C的80%,不同处理之间没有显著性差异。直至标记后的第30天,茎和叶内~(13)C的滞留量约占初始量的30%。硝态氮处理下的值在第21天和第30天显著低于对照和氨态氮处理下的值,表明硝态氮处理下,植物光合固定的碳在短期内迅速输入地下组织和土壤中。这些结果从机理上阐明了植物光合碳分配对不同化学形态氮素长期输入的响应,进而影响到土壤呼吸CO_2的释放,以及对土壤碳库动态的贡献。加深了对高寒草甸土壤有机碳库稳定性维持机制的认识,能够为高寒草地的科学管理以及资源的可持续利用提供理论指导。     

Refined Analysis of Brain Energy Metabolism Using In Vivo Dynamic Enrichment of 13C Multiplets

Carbon-13 nuclear magnetic resonance spectroscopy in combination with the infusion of ¹³C-labeled precursors is a unique approach to study in vivo brain energy metabolism. Incorporating the maximum information available from in vivo localized ¹³C spectra is of importance to get broader knowledge on cerebral metabolic pathways. Metabolic rates can be quantitatively determined from the rate of ¹³C incorporation into amino acid neurotransmitters such as glutamate and glutamine using suitable mathematical models. The time course of multiplets arising from ¹³C-¹³C coupling between adjacent carbon atoms was expected to provide additional information for metabolic modeling leading to potential improvements in the estimation of metabolic parameters.The aim of the present study was to extend two-compartment neuronal/glial modeling to include dynamics of ¹³C isotopomers available from fine structure multiplets in ¹³C spectra of glutamate and glutamine measured in vivo in rats brain at 14.1 T, termed bonded cumomer approach. Incorporating the labeling time courses of ¹³C multiplets of glutamate and glutamine resulted in elevated precision of the estimated fluxes in rat brain as well as reduced correlations between them.