A theory for 15N/14N fractionation in nitrate-grown vascular plants

We present a theory describing how the δ¹⁵N values of the nitrogen (N) pools in a vascular plant depend on that of its source N (nitrate), on ¹⁵N/¹⁴N fractionations during N assimilation, and on N transport within and N loss from the plant. The theory allows measured δ¹⁵N values to be interpreted in terms of physiological processes. The δ¹⁵N values of various N pools are calculated using three rules: (1) when a pool divides without transformation, there is no change in the δ¹⁵N values of the N entering the resulting pools; (2) when nitrate is assimilated by nitrate reductase, the δ¹⁵N values of the resulting pools (product and residual substrate) are described by a Rayleigh equation; (3) when two N pools mix, the δ¹⁵N value of the mixture is a weighted average of the δ¹⁵N values of the component pools. The theory is written as a spreadsheet and solved numerically. Potentially, it has multiple solutions. Some contravene physiological reality and are rejected. The remainder are distinguished, where possible, using additional physiological information. The theory simulated independent measurements of δ¹⁵N in N pools of Brassica campestris L. var. rapa (komatsuna) and Lycopersicon esculentum Mill. cv. T-5 (tomato). Received: 27 October 1997 / Accepted: 13 January 1998     

14C-methylamine-glutaraldehyde conjugation as an alternative to iodination for protein labeling

Radiolabeling of native proteins conventionally has required iodination using 125Iodine (125I). Although radioiodination can result in high specific activity, there are several drawbacks in the use of 125I (e.g., radiological hazards and short half-life). 14C-Methylamine-glutaraldehyde conjugation to proteins offers an alternative for radiolabeling of proteins that is safer and longer-lived alpha-2-Macroglobulin was radiolabeled by conjugation to a 14C-methylamine-glutaraldehyde conjugate. Analysis of the labeling procedure was performed using scintillation counting, gel filtration chromatography, and protein assays. The radiolabeled alpha-2-macroglobulin was activated using established protocols and tested for functional integrity using competitive binding assays in the presence of recombinant receptor associated protein, an alternative ligand for the alpha-2-macroglobulin cellular receptor. The function of alpha-2-macroglobulin was unaffected by the labeling procedure. Comparison of 14C-methylamine-labeling and iodination by Scatchard analysis yielded nonlinear plots that suggested the presence of two sets of receptors with different binding affinities but that do not show cooperativity. This technique offers an alternative to radioiodination for the sensitive labeling of proteins.     

Glycine metabolism in intact leaves by in vivo 13C and 15N labeling

Solid-state (13)C NMR measurements of intact soybean leaves labeled by (13)CO(2) (at subambient concentrations) show that excess glycine from the photorespiratory C(2) cycle (i.e. glycine not part of the production of glycerate in support of photosynthesis) is either fully decarboxylated or inserted as (13)C-labeled glycyl residues in proteins. This (13)C incorporation in leaf protein, which is uniformly (15)N labeled by (15)NH(4)(15)NO(3), occurs as soon as 2 min after the start of (13)CO(2) labeling. In those leaves with lower levels of available nitrogen (as measured by leaf nitrate and glutamine-glutamate concentrations), the excess glycine is used primarily as glycyl residues in protein.     

15N natural abundances and N use by tundra plants

Plant species collected from tundra ecosystems located along a north-south transect from central Alaska to the north coast of Alaska showed large and consistent differences in ¹⁵N natural abundances. Foliar ¹⁵N values varied by about 10% among species within each of two moist tussock tundra sites. Differences in ¹⁵N contents among species or plant groups were consistent across moist tussock tundra at several other sites and across five other tundra types at a single site. Ericaceous species had the lowest ¹⁵N values, ranging between about –8 to –6. Foliar ¹⁵N contents increased progressively in birch, willows and sedges to maximum ¹⁵N values of about +2 in sedges. Soil ¹⁵N contents in tundra ecosystems at our two most intensively studied sites increased with depth and ¹⁵N values were usually higher for soils than for plants. Isotopic fractionations during soil N transformations and possibly during plant N uptake could lead to observed differences in ¹⁵N contents among plant species and between plants and soils. Patterns of variation in ¹⁵N content among species indicate that tundra plants acquire nitrogen in extremely nutrient-poor environments by competitive partitioning of the overall N pool. Differences in plant N sources, rooting depth, mycorrhizal associations, forms of N taken up, and other factors controlling plant N uptake are possible causes of variations in ¹⁵N values of tundra plant species.     

Coupling sap flow velocity and amino acid concentrations as an alternative method to (15)N labeling for quantifying nitrogen remobilization by walnut trees

The temporal dynamics of N remobilization was studied in walnut (Juglans nigra x regia) trees growing in sand culture. Trees were fed with labeled N ((15)N) during 1999 and unlabeled N in 2000. Total N and (15)N contents in different tree compartments were measured during 80 d after bud burst and were used to estimate N remobilization for spring growth. The seasonal (and occasionally diurnal) dynamics of the concentration and (15)N enrichment of the major amino acids in xylem sap were determined concurrently. Sap flow velocity was also measured for sample trees. A new approach coupling amino acid concentrations to sap flow velocity for quantifying N remobilization was tested. A decrease of the labeled N contents of medium roots, tap roots, and trunk was observed concurrently to the increase in the labeled N content of new shoots. Remobilized N represented from previous year storage 54% of N recovered in new shoots. Arginine, citruline, gamma-amino butyric acid, glutamic acid, and aspartic acid always represented around 80% of total amino acid and amide N in xylem sap and exhibited specific seasonal trends and significant diurnal trends. N translocation was mainly insured by arginine during the first 15 d after bud burst, and then by glutamic acid and citruline. The pattern of N remobilization estimated by the new approach was consistent with that measured by the classical labeling technique. Implications for quantifying N remobilization for large, field-growing trees are discussed.     

14N and 15N imaging by SIMS microscopy in soybean leaves

The distribution of ¹⁵N and ¹⁴N compounds in cryofixed and resin embedded sections of soybean (Glycine max L) leaves was studied by SIMS microscopy. The results indicate that, with a mass resolution M/ΔM higher than 6000, images of the nitrogen distribution can be obtained from the mapping of the two secondary cluster ions ¹²C¹⁴N^? and ¹²C¹⁵N^?, in samples of both control and ¹⁵N-labeled leaves. The ionic images were clearly related to the histological structure of the organ, and allow the detection of ¹⁴N and ¹⁵N at the subcellular level. Furthermore, relative measurements of the ¹²C¹⁴N^? and ¹²C¹⁵N^? beams made possible the quantification of the ¹⁵N atom% in the various tissues of the leaf.     

Study of early leaf senescence in Arabidopsis thaliana by quantitative proteomics using reciprocal 14N/15N labeling and difference gel electrophoresis

Leaf senescence represents the final stage of leaf development and is associated with fundamental changes on the level of the proteome. For the quantitative analysis of changes in protein abundance related to early leaf senescence, we designed an elaborate double and reverse labeling strategy simultaneously employing fluorescent two-dimensional DIGE as well as metabolic (15)N labeling followed by MS. Reciprocal (14)N/(15)N labeling of entire Arabidopsis thaliana plants showed that full incorporation of (15)N into the proteins of the plant did not cause any adverse effects on development and protein expression. A direct comparison of DIGE and (15)N labeling combined with MS showed that results obtained by both quantification methods correlated well for proteins showing low to moderate regulation factors. Nano HPLC/ESI-MS/MS analysis of 21 protein spots that consistently exhibited abundance differences in nine biological replicates based on both DIGE and MS resulted in the identification of 13 distinct proteins and protein subunits that showed significant regulation in Arabidopsis mutant plants displaying advanced leaf senescence. Ribulose 1,5-bisphosphate carboxylase/oxygenase large and three of its four small subunits were found to be down-regulated, which reflects the degradation of the photosynthetic machinery during leaf senescence. Among the proteins showing higher abundance in mutant plants were several members of the glutathione S-transferase family class phi and quinone reductase. Up-regulation of these proteins fits well into the context of leaf senescence since they are generally involved in the protection of plant cells against reactive oxygen species which are increasingly generated by lipid degradation during leaf senescence. With the exception of one glutathione S-transferase isoform, none of these proteins has been linked to leaf senescence before.     

Novel approach for peptide quantitation and sequencing based on 15N and 13C metabolic labeling

Here we describe a method for protein identification and quantification using stable isotopes via in vivo metabolic labeling of the hyperthermophilic crenarchaeon Sulfolobus solfataricus. Stable isotope labeling for quantitative proteomics is becoming increasingly popular; however, its usefulness in protein identification has not been fully exploited. We use both 15N and 13C labeling to create three different versions of the same peptide, corresponding to the unlabeled, 15N and 13C labeled versions. The peptide then appears as three different peaks in a TOF-MS scan and three corresponding sets of MS/MS spectra are obtained. With this information, the elemental carbon and nitrogen compositions for each peptide and each fragment can be calculated. When this is used as a constraint in database searching and/or de novo sequencing, the confidence of a match is increased (for an example intact peptide from 34 choices to 1). This makes the method a useful proteomic tool for both sequenced and unsequenced organisms. Furthermore, it allows for accurate protein quantitation (standard deviations over >4 peptides per protein were within 10%) of three phenotypes in one MS experiment. Abundances for each peptide are calculated by determining the relative areas of each of the three peaks in the TOF-MS spectrum.     

High-level 2H/13C/15N labeling of proteins for NMR studies

Summary The protein human carbonic anhydrase II (HCA II) has been isotopically labeled with ²H, ¹³C and ¹⁵N for high-resolution NMR assignment studies and pulse sequence development. To increase the sensitivity of several key ¹H/¹³C/¹⁵N triple-resonance correlation experiments, ²H has been incorporated into HCA II in order to decrease the rates of ¹³C and ¹H_N T₂ relaxation. NMR quantities of protein with essentially complete aliphatic ²H incorporation have been obtained by growth of E. coli in defined media containing D₂O, [1,2-¹³C₂, 99%] sodium acetate, and [¹⁵N, 99%] ammonium chloride. Complete aliphatic deuterium enrichment is optimal for ¹³C and ¹⁵N backbone NMR assignment studies, since the ¹³C and ¹H_N T₂ relaxation times and, therefore, sensitivity are maximized. In addition, complete aliphatic deuteration increases both resolution and sensitivity by eliminating the differential ²H isotopic shift observed for partially deuterated CH_nD_m moieties.     

Stereospecific assignments of glycine in proteins by stereospecific deuteration and 15N labeling

Summary A method is described for stereospecifically assigning the -protons of glycine residues in proteins. The approach involves the stereospecific deuteration and ¹⁵N labeling of glycine and subsequent selective incorporation of this residue into the protein. The stereospecific assignments of the glycine -protons are obtained from a comparison of a 3D ¹⁵N-resolved TOCSY spectrum of the uniformly ¹⁵N-labeled protein with a 2D/3D ¹⁵N-edited TOCSY spectrum of the protein, containing the stereospecifically deuterated and ¹⁵N-labeled glycine. The approach is demonstrated by stereospecifically assigning the glycine -protons of the FK506 binding protein when bound to the immunosuppressant ascomycin.     

Uniform labeling of a recombinant antibody Fv-fragment with 15N and 13C for heteronuclear NMR spectroscopy

The expression of functional antibody fragments in Escherichia coli enables a detailed analysis by NMR spectroscopy. This is demonstrated with the uniform labeling of an Fv-fragment (25 kDa) comprising the antigen binding site of an antibody against 2-phenyloxazolone with 15N and 13C. The antigen-complexed Fv-fragment was analysed for a potential assignment by heteronuclear multi-dimensional NMR spectroscopy. For almost all backbone amides 15N/1H crosspeaks and for 80% of them TOCSY crosspeaks were observed. In a 13C-edited-HCCH-2D experiment 17 out of 18 threonine spin-systems were identified. Thus detailed assignments are possible, but some amino acid specific labeling in addition to uniform labeling will be required for complete assignments of Fv-fragments.     

Use of anin situ labeling technique for the determination of seasonal14C distribution in Ponderosa pine

A¹⁴C labeling apparatus was developed to permit the labeling of four-year-old Ponderosa pine with¹⁴CO₂ in the field. The labeling system is a completely closed canopy system with¹⁴CO₂ monitored by a GM tube ratemeter apparatus. The level of¹⁴CO₂ corresponding to ambient levels is monitored by a microloggercomputer which controls a¹⁴CO₂ generating system. The generated¹⁴CO₂ is mixed in the canopy by circulating the atmosphere with 12V diaphram pumps. The portable system requires little operator attention. At approximately monthly intervals over a one-year period two four-year-old Ponderosa pine trees were labeled for three to five days using this labeling apparatus. After an assimilate distribution period, one tree was excavated and analyzed for¹⁴C distribution. During late spring and early summer most of the carbon assimilated (>60%) was found in the active growing tips and new needles, with little being allocated to the roots (<10%) or woody material (<20%). During mid to late fall there was an increase in root labeling along with an increase in carbon going to woody material. Over the winter period, most of the fixed carbon (65%) resided in the older leaves. The early spring labeling period showed another pulse of root labeling along with some labeling of woody tissues.     

Box-modeling of 15N/14N in mammals

The ¹⁵N/¹⁴N signature of animal proteins is now commonly used to understand their physiology and quantify the flows of nutrient in trophic webs. These studies assume that animals are predictably ¹⁵N-enriched relative to their food, but the isotopic mechanism which accounts for this enrichment remains unknown. We developed a box model of the nitrogen isotope cycle in mammals in order to predict the ¹⁵N/¹⁴N ratios of body reservoirs as a function of time, N intake and body mass. Results of modeling show that a combination of kinetic isotope fractionation during the N transfer between amines and equilibrium fractionation related to the reversible conversion of N-amine into ammonia is required to account for the well-established ≈4‰ ¹⁵N-enrichment of body proteins relative to the diet. This isotopic enrichment observed in proteins is due to the partial recycling of ¹⁵N-enriched urea and the urinary excretion of a fraction of the strongly ¹⁵N-depleted ammonia reservoir. For a given body mass and diet δ¹⁵N, the isotopic compositions are mainly controlled by the N intake. Increase of the urea turnover combined with a decrease of the N intake lead to calculate a δ¹⁵N increase of the proteins, in agreement with the observed increase of collagen δ¹⁵N of herbivorous animals with aridity. We further show that the low δ¹⁵N collagen values of cave bears cannot be attributed to the dormancy periods as it is commonly thought, but inversely to the hyperphagia behavior. This model highlights the need for experimental investigations performed with large mammals in order to improve our understanding of natural variations of δ¹⁵N collagen.     

Direct flux and 15N tracer methods for measuring denitrification in forest soils

Estimates of denitrification are one of the key uncertainties in the terrestrial nitrogen (N) cycle, primarily because reliable measurements of this highly variable process—especially the production of its terminal product (N₂)—are difficult to obtain. We evaluated the ability of gas-flow soil core and ¹⁵N tracer methods to provide reliable estimates of denitrification in forest soils. Our objectives were to: (1) describe and present typical results from new gas-flow soil core and in situ ¹⁵N tracer methods for measuring denitrification, (2) discuss factors that affect the relevance of these methods to actual in situ denitrification, and (3) compare denitrification estimates produced by the two methods for a series of sites in a northern hardwood forest ecosystem. Both methods were able to measure accumulations of N₂ over relatively short (2–5 h) incubations of either unamended or tracer-amended intact soils. Denitrification rates measured by the direct flux soil core method were very sensitive to incubation oxygen (O₂) concentration and decreased with increased O₂ levels. Denitrification rates measured by the in situ ¹⁵N tracer method were very sensitive to the ¹⁵N content of the nitrate (NO₃ ^?) pool undergoing denitrification, which limits the applicability of this method for quantifying denitrification in N-poor ecosystems. While its ability to provide accurate estimates of denitrification was limited, the ¹⁵N tracer method provided estimates of the short-term abiotic and biotic transformations of atmospheric N deposition to gas. Furthermore, results suggest that denitrification is higher and that N₂O:N₂ ratios are lower (<0.02) than previously thought in the northern hardwood forest and that short-term abiotic and biotic transformations of atmospheric N deposition to gas are significant in this ecosystem.     

15N and 13C labeling of Escherichia coli tRNAs toward the NMR analysis.

Escherichia coli tRNAs were labeled with stable isotope 15N in vivo. Three species of tRNA, tRNA(Glu), tRNA(Lys) and tRNA(Ile), were purified by an HPLC system and their NMR spectra were observed. In heteronuclear 1H-15N multiple or single quantum coherence (HMQC or HSQC) spectra, the crosspeaks corresponding to NH3 of U and NH1 of G can be distinguished clearly since their 15N chemical shifts are significantly different from each other. Thus, this combination of 15N-labeling and the proton detected heteronuclear experiments are useful for the signal assignment and the conformational analysis of tRNAs. Furthermore, C1'- selective 13C-labeling of nucleotides was examined in vivo in order to resolve the H1' signals of tRNAs. By using a newly constructed E. coli mutant strain, the isotopic enrichments of more than 90% at C1' and of less than 10% for other ribose carbons were achieved.     

A 15N tracer technique for assessing fine root production and mortality

We tested a ¹⁵N tracer technique to assess fine root production and mortality based on temporal measurements of the ¹⁵N mass in fine root structural tissues and the ¹⁵N concentration of the plant-available soil N pool. The results of a pilot study indicated that this technique is based on sound methods and reasonable assumptions. The ¹⁵N tracer technique avoids most of the major limitations which hinder existing methods and may provide valuable insight into the rates and controls of fine root production and mortality in terrestrial ecosystems. Received: 23 September 1996 / Accepted: 10 June 1997     

Chemical influences on 14C and 15N primary production in an arctic lake

Summary Twelve 24 h bioassay experiments were conducted in 1980 and 1981 to evaluate seasonal influences of NO ₃ ^- , NH ₄ ⁺ , PO ₄ ^3- , N+P, vitamins, trace metals, a synthetic chelate and common salts on ¹⁴C and ¹⁵N primary production in Toolik Lake, Alaska. Addition of N+P, NO ₃ ^- or NH ₄ ⁺ significantly increased ¹⁴C primary production over all other treatments on most dates. Only on three occasions did any other treatment have any statistically significant effect on ¹⁴C primary production. ¹⁵NO ₃ ^- and ¹⁵NH ₄ ⁺ primary production were each significantly enhanced by PO ₄ ^3- enrichement relative to all other nutrient applications on seven dates. Significantly depressed ¹⁵NO ₃ ^- primary production consistently resulted from NH ₄ ⁺ addition but enrichment with NO ₃ ^- gave significantly depressed ¹⁵NH ₄ ⁺ primary production in just three experiments. Other treatments significantly influenced ¹⁵N primary production on two occasions only. The general stimulatory influence of N+P, NO ₃ ^- and NH ₄ ⁺ on ¹⁴C primary production as well as the similar effect of PO ₄ ^3- on ¹⁵N primary production had no seasonal pattern. The total data show that nitrogen and phosphorus are the most important chemical regulators of primary production in Toolik Lake.