Molecular recognition of adenosine deaminase: 15N NMR studies

The elucidation of the molecular recognition of adenosine deaminase (ADA), the interpretation of the catalytic mechanism, and the design of novel inhibitors are based mostly on data obtained for the crystalline state of the enzyme. To obtain evidence for molecular recognition of the physiologically relevant soluble enzyme, we studied its interactions with the in situ formed inhibitor, 6-OH-purine riboside (HDPR), by 1D-15N- and 2D-(1H-15N)- NMR using the labeled primary inhibitor [15N4]-PR. We synthesized both [15N4]-PR and an [15N4]-HDPR model, from relatively inexpensive 15N sources. The [15N4]-HDPR model was used to simulate H-bonding and possible Zn2+-coordination of HDPR with ADA. We also explored possible ionic interactions between PR and ADA by 15N-NMR monitored pH-titrations of [15N4]-PR. Finally, we investigated the [15N4]-PR-ADA 1:1 complex by 2D-(1H-15N) NMR. We found that HDPR recognition determinants in ADA do not include any ionic-interactions. HDPR N1 H is an H-bond acceptor, and not an H-bond donor. Despite the proximity of N7 to the Zn2+-ion, no coordination occurs; instead, N7 is an H-bond acceptor. We found an overall agreement between the crystallographic data for the crystallized ADA:HDPR complex and the 15N-NMR signals for the corresponding soluble complex. This finding justifies the use of ADA's crystallographic data for the design of novel inhibitors.     

1H and 15N NMR studies of protonation and hydrogen-bonding in the binding of trimethoprim to dihydrofolate reductase

The binding of trimethoprim and [1,3,2-amino-¹⁵N₃]-trimethoprim to Lactobacillus casei dihydrofolate reductase has been studied by ¹⁵N and ¹H NMR spectroscopy. ¹⁵N NMR spectra of the bound drug were obtained by using polarisation transfer pulse sequences. The ¹⁵N chemical shifts and ¹H-¹⁵N spin-coupling constants show unambiguously that the drug is protonated on N1 when bound to the enzyme.The N1-proton resonance in the complex has been assigned using the ¹⁵N-enriched molecule. The temperature-dependence of the linewidth of this resonance has been used to estimate the rate of exchange of this proton with the solvent: 160±10s^-1 at 313 K, with an activation energy of 75 (±9) kJ·mole^-1. This is considerably faster than the dissociation rate of the drug from this complex, demonstrating that there are local fluctuations in the structure of the complex.     

Synthesis, characterization and X-ray crystal structure of [Re(L4)(CO)3]Br · 2CH3OH (L4 = N,N-bis[(2-diphenylphosphino)ethyl]methoxyethylamine): A model compound for novel cationic Tc(I)-tricarbonyl radiotracers useful for heart imaging

This report describes synthesis and evaluation of cationic complexes, [^99mTc(CO)₃(L)]⁺ (L = N-methoxyethyl-N,N-bis[2-(bis(3-ethoxypropyl)phosphino)ethyl]amine (L1), N-[(15-crown-5)-2-yl]-N,N-bis[2-(bis(3-ethoxypropyl)phosphino)ethyl]amine (L2) and N-[(18-crown-6)-2-yl]-N,N-bis[2-(bis(3-ethoxypropyl)phosphino)ethyl]amine (L3)) as potential radiotracers for heart imaging. Preliminary results from biodistribution studies in female adult BALB-c mice indicated that the cationic ^99mTc(I)-tricarbonyl complex, [^99mTc(CO)₃(L2)]⁺, has a significant localization in the heart at 60 min post-injection. To understand the coordination chemistry of these bisphosphine ligands with the ^99mTc(I)-tricarbonyl core, we prepared [Re(CO)₃(L4)]Br (L4: N,N-bis[(2-diphenylphosphino)ethyl]methoxyethylamine) as a model compound. [Re(CO)₃(L4)]Br has been characterized by elemental analysis, IR, ESI-MS, NMR (¹H, ¹³C, ¹H-¹H COSY, and ¹H-¹³C HMQC) methods, and X-ray crystallography. In solid state, [Re(CO)₃(L4)]⁺ has a distorted octahedron coordination geometry with PNP occupying one facial plane. The chelator backbone adopts a “chair” conformation with phosphine-P atoms at equatorial positions and the amine-N at the apical site. In solution, [Re(CO)₃(L4)]⁺ is able to maintain its cationic nature with no dissociation of carbonyl ligands or any of the three PNP donors.     

Dihydrohexacyanoferrates of N-heterocyclic cations

Dihydrohexacyanoferrates (II and III) of aromatic N-heterocyclic cations X⁺ (such as N-methylquinoxalinium, pyridinium, dipyridinium) and X²⁺ (such as pyridylpyridinium, dipyridinium) are synthesized and characterized. For the first time, the crystal structures of acidic dihydrohexacyanoferrates are described. The formation of the and X²⁺H₂[Fe(CN)₆] species which contain the [Fe(CN)₆]⁴⁻ and [Fe(CN)₄(CNH)₂]²⁻ anions from acidic solutions occurs after the formation of the H[Fe(CN)₆]³⁻ species as can be established from the outer-sphere charge transfer (OSCT) bands in the absorption spectra. The crystal structures of these species contain extensive network of intermolecular N-H?N, N-H?O and O-H?N hydrogen bonds which link the hexacyanoferrate anions with solvent water (if present) and N-heterocyclic cations if the later can participate in the H-bond formation. In the crystals of dihydrohexacyanoferrates, the H-bond networks can be two-dimensional (species 1) and three-dimensional (species 2-7). The lack of acidic protons for the H-bond network formation can be compensated by solvent water molecules. The H-bond network plays an important role in stabilization of such strongly-acidic species such as the H₂Bpy²⁺ and HPypy²⁺ cations and the [Fe^II(CN)₄(CNH)₂]²⁻ anion.     

Up-regulation of Brain N-Methyl- -aspartate Receptors Following Multiple Intracerebro- ventricular Injections of [ -Pen, -Pen] Enkephalin and [ -Ala, Glu]deltorphin II in Mice

Bhargava, H. N., S. Kumar and J. T. Bian. Up-regulation of brain N-methyl- -aspartate receptors following multiple intracerebroventricular injections of [ -Pen², -Pen⁵]enkephalin and [ -Ala², Glu⁴]deltorphin II in mice. Peptides 18(10) 1609–1613, 1997.—The effects of chronic administration of [ -Pen², -Pen⁵]enkephalin and [ -Ala², Glu⁴]deltorphin II, the selective agonists of the δ₁- and δ₂-opioid receptors, on the binding of [³H]MK-801, a noncompetitive antagonist of the N-methyl- -aspartate receptor, were determined in several brain regions of the mouse. Male Swiss-Webster mice were injected intracerebroventricularly (i.c.v.) with [ -Pen², -Pen⁵]enkephalin or [ -Ala², Glu⁴]deltorphin II (20 μg/mouse) twice a day for 4 days. Vehicle injected mice served as controls. Previously we have shown that the above treatment results in the development of tolerance to their analgesic activity. The binding of [³H]MK-801 was determined in brain regions (cortex, midbrain, pons and medulla, hippocampus, striatum, hypothalamus and amygdala). At 5 nM concentration, the binding of [³H]MK-801 was increased in cerebral cortex, hippocampus, and pons and medulla of [ -Pen², -Pen⁵]enkephalin treated mice. In [ -Ala², Glu⁴]deltorphin II treated mice, the binding of [³H]MK-801 was increased in cerebral cortex and hippocampus. The changes in the binding were due to increases in the B_max value of [³H]MK-801. It is concluded that tolerance to δ₁- and δ₂-opioid receptor agonists is associated with up-regulation of brain N-methyl- -aspartate receptors, however, some brain areas affected differ with the two treatments. The results are consistent with the recent observation from this laboratory that N-methyl- -aspartate receptors antagonists block tolerance to the analgesic action of δ₁- and δ₂-opioid receptor agonists.     

C, N CP MAS and high resolution multinuclear NMR study of methyl

A quantitative analysis of 2D ¹H-¹⁵N spectra is often complicated by resonance overlap. Here a simple method is presented for resolving overlapped correlations by recording 2D projection planes from HNCO data sets. Applications are presented involving the measurement of ¹⁵N T₁ relaxation rates in a high molecular weight protein, malate synthase G, and in a system that exchanges between folded and unfolded states, the drkN SH3 domain. By supplementing relaxation data recorded in the conventional way as a series of 2D ¹H-¹⁵N data sets with a series of a pair of projection planes the number of dynamics probes is increased significantly for both systems studied.     

Addressing the overlap problem in the quantitative analysis of two dimensional NMR spectra: Application to <Superscript>15</Superscript>N relaxation measurements

A quantitative analysis of 2D ¹H-¹⁵N spectra is often complicated by resonance overlap. Here a simple method is presented for resolving overlapped correlations by recording 2D projection planes from HNCO data sets. Applications are presented involving the measurement of ¹⁵N T₁ relaxation rates in a high molecular weight protein, malate synthase G, and in a system that exchanges between folded and unfolded states, the drkN SH3 domain. By supplementing relaxation data recorded in the conventional way as a series of 2D ¹H-¹⁵N data sets with a series of a pair of projection planes the number of dynamics probes is increased significantly for both systems studied.     

15N resonance assignments of oxidized and reducedChromatium vinosum high-potential iron protein

The¹⁵N resonances in reduced and oxidizedChromatium vinosum high-potential iron protein have been assigned by use of¹H-¹H COSY spectra and¹H-¹⁵N HMQC, HMQC-COSY, and HMQC-NOESY spectra. Unambiguous assignment of 70 of 85 backbone¹⁵N resonances in the reduced protein and 62 of 85 resonances in the oxidized protein are made, as are 12 of 21 side-chain¹⁵N resonances.     

Body nitrosation potential measured by a novel N breath test

Oxygenated nitrogen species, for example, the protonated form of nitrous acid (H₂ONO⁺), dinitrogentrioxide (N₂O₃), dinitrogentetroxide (N_2O4), or peroxynitrite (ONOO⁻), can react with amines to form molecular nitrogen. These reactions can occur spontaneously with primary aliphatic amines or via cytochrome P450 catalysed reactions with secondary amines. In principle measurements of the excretion of the molecular nitrogen generated by these reactions could be used as an index of the levels of oxygenated nitrogen compounds acting as nitrosating agents. To test this idea, [¹⁵N₂]urea (3 mmol) was administered orally to five patients infected with Helicobacter pylori (as diagnosed by the [¹³C]urea breath test) and to four healthy volunteers. All participants ingested 3-mmol sodium nitrate as a precursor for NA 5 min before the ingestion of the nitrogen tracer. During the test the participants breathed 100% oxygen to increase the sensitivity of detection of endogenous molecular nitrogen. After the administration of [¹⁵N₂]urea, the patients with H. pylori showed significantly increased ¹⁵N enrichments of exhaled N₂, expressed as δ value (‰), compared with healthy volunteers (patients: 3.5 ± 0.9 vs. volunteers: 1.3 ± 0.4; p < .05). We speculate that the endogenous production of molecular nitrogen is a protective process controlling the body NO and nitrite levels. The ¹⁵N breath technique allows the noninvasive estimation of the body nitrosation and could indicate the health risk, possibly the oxidative stress status, caused by highly reactive oxygenated nitrogen species and carbenium ion intermediates.     

N-NMR characterization of His residues in and around the active site of FeSOD

We have exploited ¹⁵N-NMR to observe histidine (His) side chains in and around the active site of Fe-containing superoxide dismutase (FeSOD). In the oxidized state, we observe all the non-ligand His side chains and in the reduced state we can account for all the signals in the imidazole spectral region in terms of the non-ligand His′, paramagnetically displaced signals from two backbone amides, and the side chain of glutamine 69 (Gln69). We also observe signals from the His′ that ligate Fe^II. These confirm that neither the Q69H nor the Q69E mutation strongly affects the Fe^II electronic structure, despite the 250 mV and > 660 mV increases in E_m they produce, respectively. In the Q69H mutant, we observe two new signals attributable to the His introduced into the active site in place of Gln69. One corresponds to a protonated N and the other is strongly paramagnetically shifted, to 500 ppm. The strong paramagnetic effects support the existence of an H-bond between His69 and the solvent molecule coordinated to Fe^II, as proposed based on crystallography. Based on previous information that His69 is neutral, we infer that the shifted N is not protonated. Therefore, we propose that this N represents a site of H-bond acceptance from coordinated solvent, representing a reversal of the polarity of this H-bond from that in WT (wild-type) FeSOD protein. We also present evidence that substrate analogs bind to Fe^IISOD outside the Fe^II coordination sphere, affecting Gln69 but without direct involvement of His30.     

Conformationally Restricted 2-Substituted Wyosine Derivatives. 1H, 13C,and 15N NMR Study

Abstract

It was found by ¹H, ¹³C and ¹⁵N NMR study that substitution of 4,9-dihydro-4, 6-dimethyl-9-oxo-3-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl) imidazo [1,2-a]purine (wyosine triacetate, 1) at C2 position with electronegative groups CH₃O and C₆H₅CH₂O results in a noticeable electron distribution disturbance in the “left-hand” imidazole ring and a significant increase in the North conformer population of the sugar moiety.     

Synthesis and characterization of two new triazolate-aliphatic dicarboxylate bridged Zn(II) coordination polymers based on 2D layer motifs with different crystal packing

Two new zinc(II)-triazole-aliphatic dicarboxylate coordination polymers, [Zn(trz)(Hsuc)]_n (1), [Zn₂(trz)₂(tar)]_n (2), have been hydrothermally synthesized by reaction of Zn salt, Htrz with H₂suc and H₂tar, respectively (Htrz = 1,2,4-triazole, H₂suc = succinic acid, H₂tar = tartaric acid).Their structures were determined by single-crystal X-ray diffraction analyses and further characterized by X-ray powder diffraction, elemental analyses, IR spectra and TG analyses. Compound 1 displays a 2D layer structure containing {[Zn₄(trz)₄]⁴⁺}_n layers decorated by the suc ligand. Compound 2 is in a 3D structure formed by the interconnection of 2D {[Zn₄(trz)₄]⁴⁺}_n layers with tar ligand, resulting a 3,4-connected topological network. Due to the different coordination mode and conformation of aliphatic carboxylate ligand, the similar 2D {[Zn₄(trz)₄]⁴⁺}_n layers stack in the -AAA- fashion in 1, while the {[Zn₄(trz)₄]⁴⁺}_n layers hold together in the -ABAB- stacking sequence in 2. Additionally, the two compounds show strong fluorescence in the solid state at room temperature.     

Synthesis and characterization of gold(III) complexes with alkyldiamine ligands

A series of gold(III) metalacycle of five-, six- and seven-membered ring was prepared by reacting Auric acid (HAuCl₄ · 3H₂O) with 1 equiv. unsubstituted ethylenediamine (en), propylene diamine (pn) and butylenediamine (bn) ligands and with some N-mono-substituted as well as N,N′-disubstituted ethylenediamine ligands. The general formula of these complexes is [Au(alkyldiamine)Cl₂]Cl. These complexes are characterized by melting point and elemental analysis, while structural analysis was done by spectroscopic techniques such as UV-Vis, Far-IR, IR spectroscopy, ¹H and ¹³C solution as well as ¹³C and ¹⁵ N solid-state NMR. The solid-state ¹⁵ N NMR shows that the chemical shift difference between free and bound ligand decreases as bn > pn > en, indicating stronger Au-N bond for bn complex compared to pn and en. UV-Vis shows relative stability of the Au(III) complexes of unsubstituted ethylenediamine with respect to N,N′-di-substituted ethylenediamine. Far-IR data show the six-membered metalacycle gold(III) alkanediamine complexes to be more stable. Spectroscopic data are evaluated by comparisons with calculated data of the built and optimized structure by gaussian03 at the RB3LYP level with LanL2DZ bases set.     

Dinitrogen-fixation by three neotropical agroforestry tree species under semi-controlled field conditions

Cultivating dinitrogen-fixing legume trees with crops in agroforestry is a relatively common N management practice in the Neotropics. The objective of this study was to assess the N₂ fixation potential of three important Neotropical agroforestry tree species, Erythrina poeppigiana, Erythrina fusca, and Inga edulis, under semi-controlled field conditions. The study was conducted in the humid tropical climate of the Caribbean coastal plain of Costa Rica. In 2002, seedlings of I. edulis and Vochysia guatemalensis were planted in one-meter-deep open-ended plastic cylinders buried in soil within hedgerows of the same species. Overall tree spacing was 1 × 4 m to simulate a typical alley-cropping design. The ¹⁵N was applied as (NH₄)₂SO₄ at 10% ¹⁵N atom excess 15 days after planting at the rate of 20 kg [N] ha⁻¹. In 2003, seedlings of E. poeppigiana, E. fusca, and V. guatemalensis were planted in the same field using the existing cylinders. The ¹⁵N application was repeated at the rate of 20 kg [N] ha⁻¹ 15 days after planting and 10 kg [N] ha⁻¹ was added three months after planting. Trees were harvested 9 months after planting in both years. The ¹⁵N content of leaves, branches, stems, and roots was determined by mass spectrometry. The percentage of atmospheric N fixed out of total N (%N_f) was calculated based on ¹⁵N atom excess in leaves or total biomass. The difference between the two calculation methods was insignificant for all species. Sixty percent of I. edulis trees fixed N₂; %N_f was 57% for the N₂-fixing trees. Biomass production and N yield were similar in N₂-fixing and non-N₂-fixing I. edulis. No obvious cause was found for why not all I. edulis trees fixed N₂. All E. poeppigiana and E. fusca trees fixed N₂; %N_f was ca. 59% and 64%, respectively. These data were extrapolated to typical agroforestry systems using published data on N recycling by the studied species. Inga edulis may recycle ca. 100 kg ha⁻¹ a⁻¹ of N fixed from atmosphere to soil if only 60% of trees fix N₂, E. poeppigiana 60–160 kg ha⁻¹ a⁻¹, and E. fusca ca. 80 kg ha⁻¹ a⁻¹.     

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.     

Inhibition of GABAA ligand-gated Cl− channels by zinc in adult rat brain: A regional study

Zinc (Zn²⁺) was shown to invariably inhibit muscimol-stimulated³⁶Cl⁻ uptake by synaptoneurosomes in the cerebral cortex, hippocampus and cerebellum. The Zn²⁺ sensitivity of the GABA_A receptor-gated³⁶Cl⁻ uptake in the cerebral cortex was comparable to that in the hippocampus, whereas the uptake in the cerebellum was less sensitive to Zn²⁺. Although diazepam-potentiation of muscimol-stimulated³⁶Cl⁻ uptake was unaltered by 100 μM Zn²⁺ in the cerebellum. Zn²⁺ inhibited [³H]diazepam binding significantly at 1 mM in the cerebral cortex and cerebellum, whereas Ni²⁺ increased the binding in a concentration-dependent manner in both regions. Although lower concentrations of Zn²⁺ did not affect [³H]Ro 15-4513 binding to diazepam-sensitive sites, higher concentrations of Zn²⁺ increased the binding in both regions. Unlike the diazepam-sensitive sites the diazepam-insensitive [³H]Ro 15-4513 binding was not affected by Zn²⁺ or Ni²⁺ at any of the tested concentrations. These results suggest that the GABA_A ligand-gated Cl⁻ flux and its diazepam-potentiation are heterogeneously modulated in various brain regions. It is also suggested that cerebellar diazepam-insensitive [³H]Ro 15-4513 binding sites are insensitive to Zn²⁺ and Ni²⁺.     

Synthesis, spectroscopical characterization and the biological activity in vitro of new platinum(II) complexes with imidazo[1,5-a]-1,3,5-triazine derivatives and dimethylsulfoxide

A series of platinum(II) complexes with 6,8-dimethylimidazo[1,5-a]-1,3,5-triazin-4(3H)-one (6,8-DiMe-4-O-IMT) (I) and 6,8-dimethyl-2-thioxo-2,3-dihydroimidazo[1,5-a]-1,3,5-triazin-4(1H)-one (6,8-DiMe-4-O-2-S-IMT) (II) of formula trans-[PtCl₂(dmso)(6,8-DiMe-4-O-IMT)] (1a) and trans-[PtCl₂(dmso)(6,8-DiMe-4-O-2-S-IMT)] (2a) have been prepared and characterized with ¹H, ¹³C, ¹⁵N, ¹⁹⁵Pt NMR and IR. Significant ¹⁵N NMR upfield coordination shifts (81-96 ppm) of N(7) atom indicate this nitrogen atom as a coordination site. The multinuclear NMR and IR spectra indicate the square planar geometry with N(7) bonded heterocycles, S-bonded dimethylsulfoxide and two trans chloride anions. The platinum(II) complexes were tested for their antiproliferative activity in vitro against the cells of four human cell lines: SW707 rectal adenocarcinoma, A549 non-small cell lung carcinoma, T47D breast cancer and HCV29T bladder cancer. The activity of (1a, 2a) was lower than that of cisplatin.     

Methylmercury-Induced Elevations in Intrasynaptosomal Zinc Concentrations: An 19F-NMR Study

Abstract: Methylmercury (MeHg) increases the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) and another endogenous polyvalent cation in both synaptosomes and NG108-15 cells. In synaptosomes, the elevation in [Ca²⁺]_i was strictly dependent on extracellular Ca²⁺ (Ca²⁺_e); similarly, in NG108-15 cells, a component of the elevations in [Ca²⁺]_i was Ca²⁺_e dependent. The MeHg-induced elevations in endogenous polyvalent cation concentration were independent of Ca²⁺_e in synaptosomes and NG108-15 cells. The pattern of alterations in fura-2 fluorescence suggested the endogenous polyvalent cation may be Zn²⁺. Using ¹⁹F-NMR spectroscopy of rat cortical synaptosomes loaded with the fluorinated chelator 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5F-BAPTA), we have determined unambiguously that MeHg increases the free intrasynaptosomal Zn²⁺ concentration ([Zn²⁺]_i). In buffer containing 200 µM EGTA to prevent the Ca²⁺_e-dependent elevations in [Ca²⁺]_i, the [Zn²⁺]_i was 1.37 ± 0.20 nM; following a 40-min exposure to MeHg-free buffer [Zn²⁺]_i was 1.88 ± 0.53 nM. Treatment of synaptosomes for 40 min with 125 µM MeHg yielded [Zn²⁺]_i of 2.69 ± 0.55 nM, whereas 250 µM MeHg significantly elevated [Zn²⁺]_i to 3.99 ± 0.68 nM. No Zn²⁺ peak was observed in synaptosomes treated with the cell-permeant heavy metal chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, 100 µM) following 250 µM MeHg exposure. [Ca²⁺]_i in buffer containing 200 µM EGTA was 338 ± 26 nM and was 370 ± 64 nM following an additional 40-min exposure to MeHg-free buffer. [Ca²⁺]_i was 498 ± 28 or 492 ± 53 nM during a 40-min exposure to 125 or 250 µM MeHg, respectively. None of the values of [Ca²⁺]_i differed significantly from either pretreatment levels or buffer-treated controls.     

Nitrogen gas (N2+N2O) flux from urea applied to lowland rice as affected by green manure

A field study was conducted on a clay soil (Andaqueptic Haplaquoll) in the Philippines to directly measure the evolution of (N₂+N₂O)−¹⁵N from 98 atom %¹⁵N-labeled urea broadcast at 29 kg N ha⁻¹ into 0.05-m-deep floodwater at 15 days after transplanting (DT) rice. The flux of (N₂+N₂O)−¹⁵N during the 19 days following urea application never exceeded 28 g N ha⁻¹ day⁻¹. The total recovery of (N₂+N₂O)−¹⁵N evolved from the field was only 0.51% of the applied N, whereas total gaseous¹⁵N loss estimated from unrecovered¹⁵N in the¹⁵N balance was 41% of the applied N. Floodwater (nitrate+nitrite)−N in the 5 days following urea application never exceeded 0.14 g N m⁻³ or 0.3% of the applied N. Prior cropping of cowpea [Vigna unguiculata (L.) Walp.] to flowering with subsequent incorporation of the green manure (dry matter=2.5 Mg ha⁻¹, C/N=15) at 15 days before rice transplanting had no effect on fate of urea applied to rice at 15 DT. The recovery of (N₂+N₂O)−¹⁵N and total¹⁵N loss during the 19 days following urea application were 0.46 and 40%, respectively. Direct recovery of evolved (N₂+N₂O)−¹⁵N and total¹⁵N loss from 27 kg applied nitrate-N ha⁻¹ were 20% and 53% during the same 19-day period. The failure of directly-recovered (N₂+N₂O)−¹⁵N to match total¹⁵N loss from added nitrate-¹⁵N might be due to entrapment of denitrification end products in soil or transport of gaseous end products to the atmosphere through rice plants. The rapid conversion of added nitrate-N to (N₂+N₂O)−N, the apparently sufficient water soluble soil organic C for denitrification (101 μg C g⁻¹ in the top 0.15-m soil layer), and the low floodwater nitrate following urea application suggested that denitrification loss from urea was controlled by supply of nitrate rather than by availability of organic C.     

Effects of N-deficiency and timing of N supply on the recovery and distribution of labeled 15N in contrasting maize hybrids

Little information exists on the pattern of nitrogen (N) uptake, remobilization and N use efficiency (NUE) in Leafy and stay-green (SG) maize (Zea mays L.) genotypes. A pot experiment was conducted under controlled nutrition and growing conditions to determine the response of Leafy and SG maize genotypes to different levels of N-deficiency and timing of N supply. Three contrasting maize hybrids, Pioneer 3905 (a conventional hybrid with moderate SG characteristics), Pioneer 39F06 Bt (with a high score of SG trait) and Maizex LF850-RR (with a Leafy trait) were grown in 6 L plastic pots. Five different N treatments [no supply of N until V8 (N₁), no supply of N after V8 (N₂), no supply of N after silking (N₃), no supply of N beyond 3 weeks after silking (N₄), and continuous N supply from emergence to physiological maturity (N₅; standard check)] were imposed through modified Hoagland solution applied manually. Labeled ¹⁵N of 5% ¹⁵N₂–NH₄NO₃fertilizer was applied at 3 g per pot at the start of each schedule N treatment. Total amounts of N applied in different treatments were 3.13, 1.32, 1.90, 2.63 and 3.40 g, respectively in N₁, N₂, N₃, N₄ and N₅. Dry matter, N concentration, ¹⁵N (atom% enrichment) and NUE were determined in roots, stalk, leaves and grains at crop maturity. The three contrasting hybrids did not differ in grain yield, total N acquisition, partitioning of ¹⁵N and NUE. Restriction of N supply until V8, and from V8 to physiological maturity significantly reduced grain yield and N-uptake in all hybrids. Irrespective of the level of N-deficiency in plant and timing when the labeled fertilizer was applied, the amount of ¹⁵N recovered in the matured plant was the same in all N treatments. It has been evident that maize continued to take up N beyond 3 weeks after silking and the later N was applied during the development, the higher proportion of it was partitioned to grains. Of the total ¹⁵N uptake, 78% was partitioned to kernels in the N₄ treatment compared to only 61% in the control. Our data showed no evidence of differential N uptake, remobilization and NUE in the SG or Leafy hybrids tested, but the timing of N application and level of N-deficiency in plant significantly influenced N uptake, remobilization and N-dynamics in maize.