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.     

Studies on in vitro antipyrine metabolism by 13C,15N double labeled method

The capacities of forms of cytochrome P-450 to oxidize antipyrine were compared. An isotope dilution gas chromatography/mass spectrometry/selected ion monitoring assay was developed to quantify the three main metabolites, norantipyrine, 3-hydroxymethylantipyrine and 4-hydroxyantipyrine. 13C,15N-Double labeled antipyrine was used as a substrate and the metabolites were analyzed as their trimethylsilyl derivatives. Among forms of cytochrome P-450 examined, a male-specific form of P-450, namely P-450-male, showed higher activity to form all the three metabolites. The other forms were responsible only for the formation of norantipyrine and 4-hydroxyantipyrine. The activities of liver microsomes from untreated male and female rats and rats treated with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyl were expressed dependent on the activities of forms of cytochrome P-450 examined.     

Biosynthesis of avenic acid A in oat cv. Onward: studies with 14C or 15N labeled compounds

The precursory role of avenic acid A (AVA) in the biosynthesis of the mugineic acid family (MAs) of phytosiderophores was studied by feeding ¹⁴C or ¹⁵N labeled compounds into iron-deficient oat roots (Avena sativa L. cv. Onward). Carbon-14 of methionine was incorporated into AVA and 2-deoxymugineic acid (DMA) in the oat roots, while ¹⁴C of homoserine was not incorporated into either AVA or DMA. The molar radioactivity of DMA was higher than that of AVA. Incorporation of ¹⁵N into MAs was examined by feeding ¹⁵N-ammonium sulfate into oat roots. The value of ¹⁵N atom-% excess of DMA was higher than that of AVA.These results indicate that methionine, rather than homoserine, is the direct precursor of MAs in oat, which is similar to that in barley, and that AVA is not the precursor of the other MAs.     

Multiple-quantum HCN-CCH-TOCSY experiment for 13C/15N labeled RNA oligonucleotides

A multiple-quantum 3D HCN-CCH-TOCSY experiment is presented for the assignment of RNA ribose resonances. The experiment makes use of the chemical shift dispersion of N1 of pyrimidine and N9 of purine to distinguish the ribose spin systems. It provides an alternative approach for the assignment of ribose resonances to the currently used COSY- and TOCSY-type experiments in which either ¹³C or ¹H is utilized to distinguish the different spin systems. Compared to the single-quantum version, the sensitivity of the multiple-quantum HCN-CCH-TOCSY experiment is enhanced on average by a factor of 2 for a 23-mer RNA aptamer complexed with neomycin.     

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.     

Uptake of 13C and 15N (ammonium, nitrate and urea) by Microcystis in Lake Kasumigaura

The uptake rate of carbon and nitrogen (ammonium, nitrate andurea) by the Microcystis predominating among phytoplankton wasinvestigated in the summer of 1984 in Takahamaira Bay of LakeKasumigaura. The V_max values of Microcystis for nitrate (0.025–0.046h^–1) and ammonium (0.15–0.17 h^–1) were considerablyhigher than other natural phytoplankton. The ammonium, nitrateand urea uptake by Microcystis was light dependent and was notinhibited with nigh light intensity. The K₁ values were farlower than the I_k values. The carbon uptake was not influencedby nitrogen enrichment. Microcystis accelerated the uptake rateby changing V_max/K _s value when nitrogen versus carbon contentin cells declined. Nitrate was scarcely existent in TakahamairiBay during the summer, when Microcystis usually used ammoniumas the nitrogen source. However, the standing stock of ammoniumin the water was far lower than the daily ammonium uptake rates.Therefore, the ammonium in this water had to be supplied becauseof its rapid turn-over time (–0.7–2.6 h).     

Resonance assignment of 13C/15N labeled solid proteins by two- and three-dimensional magic-angle-spinning NMR

The comprehensive structure determination of isotopically labeled proteins by solid-state NMR requires sequence-specific assignment of ¹³C and ¹⁵ N spectra. We describe several 2D and 3D MAS correlation techniques for resonance assignment and apply them, at 7.0 Tesla, to ¹³C and ¹⁵N labeled ubiquitin to examine the extent of resonance assignments in the solid state. Both interresidue and intraresidue assignments of the ¹³C and ¹⁵N resonances are addressed. The interresidue assignment was carried out by an N(CO)CA technique, which yields N_i-C_i–1 connectivities in protein backbones via two steps of dipolar-mediated coherence transfer. The intraresidue connectivities were obtained from a new 3D NCACB technique, which utilizes the well resolved C chemical shift to distinguish the different amino acids. Additional amino acid type assignment was provided by a ¹³C spin diffusion experiment, which exhibits ¹³C spin pairs as off-diagonal intensities in the 2D spectrum. To better resolve carbons with similar chemical shifts, we also performed a dipolar-mediated INADEQUATE experiment. By cross-referencing these spectra and exploiting the selective and extensive ¹³ C labeling approach, we assigned 25% of the amino acids in ubiquitin sequence-specifically and 47% of the residues to the amino acid types. The sensitivity and resolution of these experiments are evaluated, especially in the context of the selective and extensive ¹³C labeling approach.     

Time-saving methods for heteronuclear multidimensional NMR of (13C, 15N) doubly labeled proteins

Summary Heteronuclear 2D (¹³C, ¹H) and (¹⁵N, ¹H) correlation spectra of (¹³C, ¹⁵N) fully enriched proteins can be acquired simultaneously with virtually no sensitivity loss or increase in artefact levels. Three pulse sequences are described, for 2D time-shared or TS-HSQC, 2D TS-HMQC and 2D TS-HSMQC spectra, respectively. Independent spectral widths can be sampled for both heteronuclei. The sequences can be greatly improved by combining them with field-gradient methods. By applying the sequences to 3D and 4D NMR spectroscopy, considerable time savings can be obtained. The method is demonstrated for the 18 kDa HU protein.Abbreviations HMQC heteronuclear multiple-quantum coherence spectroscopy - HSQC heteronuclear single-quantum coherence spectroscopy - HSMQC heteronuclear single- and multiple-quantum coherence spectroscopy - NOESY nuclear Overhauser enhancement spectroscopy     

Adventitions shoot formation on excised leaves of in vitro grown shoots of apple cultivars

Leaves taken from micropropagated shoots of several apple (Malus domestica Borkh.) cultivars were cultured in vitro on Linsmaier & Skoog (LS) medium or the rice anther culture medium of Chu et al. (N6) containing various concentrations of either benzyladenine (BA) or thidiazuron (TDZ) plus naphthaleneacetic acid (NAA). Of the TDZ concentrations tested, 10 M was most effective and it was equivalent to, or better than, 22 M BA for both the percentage of leaves regenerating shoots and number of shoots formed per regenerating leaf in almost every experiment. Lower concentrations of NAA (1.1 and 5.4 M) gave best results with both BA and TDZ. N6 medium gave consistently better results than LS. Lowering total salt concentration or total N concentration of LS to that of N6 did not improve the response nor did changing the NO₃:NH₄ ratio. The 3–4 leaves on the most distal part of the shoot were most responsive and tended to form the most adventitious shoots. Placing the leaf cultures in the dark for the first 2–3 weeks of the culture period produced the best results. Optimum results were obtained by culturing leaves from the distal part of the shoot in the dark for 2 weeks on N6 medium containing 10 M TDZ and 1.1 or 5.4 M NAA, then moving the cultures to 16 h daylight at a photon flux of 60 mol s^-1m^-2.     

Sensitivity optimized HCN and HCNCH experiments for 13C/15N labeled oligonucleotides

Triple resonance HCN and HCNCH experiments used in studies of ¹³C/¹⁵N labeled oligonucleotides include extended evolution periods (typically up to 100 ms) to allow coherence transfer through a complex heteronuclear spin network. Unfortunately, most of the magnetization is lost during the evolution due to fast spin–spin relaxation dominated by one-bond ¹H–¹³C dipolar interaction. As demonstrated recently, the sensitivity of the experiments can be dramatically improved by keeping the spin system in a state of proton–carbon multiple-quantum coherence, which is not affected by the strong dipolar coupling. However, the multiple-quantum coherence is very sensitive to homonuclear as well as long-range heteronuclear interactions. Unwanted magnetization transfer due to these interactions can reduce the sensitivity back to the level of a single-quantum experiment and, for some spin moieties, even eliminate the signal completely. In the present paper we show that a modified HCN scheme that refocuses the interfering coherences improves sensitivity routinely by a factor of 1.5 to 4 over a nonselective experiment. In addition, novel multiple-quantum 2D and 3D HCNCH experiments with substantially enhanced sensitivity are presented.     

Alley cropping with Senna siamea in South-western Nigeria: I. Recovery of 15N labeled urea by the alley cropping system

Improved cropping systems with in-situ production of organic matter require the input of additional inorganic N to maintain crop production in a sustainable way. For proper management of this fertilizer-N, it is necessary to quantify how the applied fertilizer N is used by the various components of the system and by the system as a whole. The fate of a single application of ¹⁵N labeled urea-N through the different components (crop, hedgerow, surface litter, soil profile up to 150 cm) of a Senna siamea alley cropping system, intercropped with maize in the first and cowpea in the second season, was followed for a period of 1.5 years (1994–1995), equivalent to 2 maize and 1 cowpea crop. Special attention was given to the role of the particulate organic matter (POM) in the cycling of urea-N through the soil organic matter (SOM). The maize crop recovered 26.5 and 1.7% of the applied urea-N at harvest in 1994 and 1995, respectively. The cowpea pods recovered only 0.7% of the applied urea-N at harvest. The highest proportion of applied urea-N recovered by the hedgerow occurred at 38 days after 1994 maize planting (DAP) (3.8%), while at later dates, recoveries of applied urea-N were always below 1%. This indicates that the Senna hedge is not a strong competitor for the applied urea-N during crop growth, i.e. while the Senna canopy is pruned at regular intervals. At 21 DAP, 12.7% of the applied urea-N was recovered in the surface litter and this value dropped significantly to 1.6% at 107 DAP and remained below 1% up to 480 DAP. The top 10 cm of soil contained 21% of the applied urea-N at 21 DAP and this value dropped to 9% at 480 DAP. Significantly more urea-N was recovered in the top 10 cm of soil than in the deeper soil layers at all sampling times. At 21 DAP, 11% of the applied urea-N was recovered in the 120–150 cm layer. This fast movement of urea-derived N to deep soil layers must have happened by preferential flow in macropores as the rainfall between urea application and the first sampling (74.2 mm) was not high enough to explain downward movement of N with the mobile water. Significant linear relationships between the proportion of urea-N in the different soil layers (excluding 0–10 cm) and the anion exchange capacity (AEC) and silt+clay content of the respective layers were found at 67, 107, 347 and 480 DAP. The total N content of the POM fraction increased significantly between 0 and 101 DAP from 127 to 171 mg N kg^–1 and decreased to 92 mg N kg^–1 at 480 DAP. The highest recovery of applied urea-N in the POM pool was measured at 101 DAP (3.6%) and this value decreased to 1.8% at 480 DAP. The total recovery of applied urea-N was 81% at 21 DAP, and decreased to values varying between 53 and 60% up from 38 to 347 DAP. At 480 DAP, the recovery decreased further to 47%. The fast movement of a substantial amount of urea-N may be responsible for this incomplete recovery, already at 21 DAP. Although the soil N status in the fertilized alley cropping system appears to be favourable for plant growth, this may be short-lived in the absence of further urea additions, as the soil-derived maize uptake in 1995 was already significantly lower than in 1994, and as the labile POM pool decreased significantly between the maize harvest in 1994 and 1995.     

Relative concentrations of N15 in urinary ammonia N and urea N after feeding N15-labeled compounds

Available data on the isotopic ratio See PDF for Equation of ammonia (r_a) and that of urea (r_u) after a single feeding of glycine, aspartic acid, and ammonium citrate are analyzed. From this analysis the following conclusions are drawn. 1. The isotopic ratio See PDF for Equation of ammonia (r_a) is always higher than that of urea (r_u) in the initial period after a single feeding of isotopic glycine or aspartic acid, but the relation is reversed later. A similar relation probably holds after feeding isotopic ammonia. 2. It is pointed out that the ratio of average r_a to average r_u depends on the time interval for which urine is collected, on the schedule of feeding, and probably also on the amount taken at each feeding. When the amount fed and the feeding schedule are unknown, theoretical interpretation of the ratio of average r_u to average r_u is impossible. 3. At the point of maximum isotopic ratio of urea, it is very probably equal to the isotopic ratio of ammonia. A possible explanation is suggested.     

Assimilation of 15N2 and 15NO3- by Partially Nitrate-Tolerant Nodulation Mutants of Soybean

Growth-chamber studies were conducted to evaluate nitrogen assimilationby three hypernodulated soybean [Glycine max (L.) Merr.] mutants(NOD1–3, NOD2–4, NOD3–7) and the Williamsparent. Seeds were inoculated at planting and transplanted atday 7 to nutrient solution with 1 mol m^–3 urea (optimizesnodule formation) or 5 mol m^–3 NO₃^– (inhibits noduleformation). At 25 d after planting, separate plants were exposedto ¹⁵NO₂ or ¹⁵NO₃^– for 3 to 48 h to evaluate N₂ fixationand NO₃^– assimilation. Plant growth was less for hypernodulatedmutants than for Williams with both NO₃^– and urea nutrition.The major portion of symbiotically fixed ¹⁵N was rapidly assimilated(30 min) into an ethanol-soluble fraction, but by 24 h aftertreatment the ethanolinsoluble fraction in each plant part wasmost strongly labelled. Distribution patterns of ¹⁵N among organswere very similar among lines for both N growth treatments aftera 24 h ¹⁵N₂ fixation period; approximate distributions were40% in nodules, 12% in roots, 14% in stems, and 34% in leaves.With urea-grown plants the totalmg ¹⁵N fixed plant^–1 24h^–1 was 1·18 (Williams), 1·40 (N0D1-3),107 (NOD2-4), and 0·80 (NOD3-7). The 5 mol m^-3 NO₃^- treatmentresulted in a 95 to 97% decrease in nodule mass and ¹⁵N₂ fixationby Williams, while the three mutants retained 30 to 40% of thenodule mass and 17 to 19% of the ¹⁵N₂ fixation of respectiveurea-grown controls. The hypernodulated mutants, which had restrictedroot growth, absorbed less ¹⁵NO₃^- than Williams, irrespectiveof prior N growthcondition. The ¹⁵N from ¹⁵NO₃^- was primarilyretained in the soluble fraction of all plant parts through24 h. The ¹⁵N incorporation studies confirmed that nodule developmentis less sensitive to external NO₃^- in mutant lines than in theWilliams parent, and provide evidence that subsequent metabolismand distribution within the plant was not different among lines.These results further confirm that the hypernodulated mutantsof Williams are similar in many respects to the hyper- or supernodulatedmutants in the Bragg background, and suggest that a common mutationalevent affectingautoregulatory control of nodulation has beentargeted. Key words: Glycine max (L.) Merr., soybean, N₂fixation, nitrate assimilation, nodulation mutants, ¹⁵N isotope