15N Application Research Articles

Root activity patterns in an Amazonian agroforest with fruit trees determined by 32P, 33P and 15N applications

In a multi-strata agroforestry system in the central Amazon near Manaus, we studied the root activity distribution of different fruit trees and a legume cover crop in comparison to monocultures and a secondary forest site. Uptake of applied 32P, 33P and 15N from 0.1, 0.6 and 1.5 m depth was compared in both the dry and wet season. The results obtained with 32P were similar to those with 15N but showed a higher variability, probably due to the lower mobility of P than N in soil and thus the labeling of a smaller soil volume with 32P. During the dry season, topsoil root activity measured with 15N was around 80% for all species with the exception of the palm tree Bactris gasipaes Kunth., which had a higher uptake from 0.6 m (50%) than from 0.1 m (30%). The subsoil (1.5 m) root activity was higher, when Bactris was not regularly cut for heart of palm harvest but grown for fruit production. Additionally, relative subsoil root activity of Theobroma increased and topsoil root activity of both Bactris and Theobroma decreased when intercropped in comparison to the monoculture. During the rainy season, the topsoil tree root activity slightly increased attributable to increasing water availability near the soil surface. The lowest isotope enrichment was noted for the secondary forest trees despite their low above ground biomass. The magnitude of the isotope enrichment was related to the foliar P and less pronounced to the foliar N contents, indicating higher nutrient cycling for nutrient-rich plant species. Despite the significant subsoil root activity (1.5 m) there was little evidence that large amounts of nutrients below 1 m depth can be recycled by the investigated tree species. More important may be a rapid recycling of nutrients from 0–1 m depth.

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.

Alley cropping with Senna siamea in South-western Nigeria: II. Dry matter, total N, and urea-derived N dynamics of the Senna and maize roots

Crop and tree roots are crucial in the nutrient recycling hypotheses related to alley cropping systems. At the same time, they are the least understood components of these systems. The biomass, total N content and urea-derived N content of the Senna and maize roots in a Senna-maize alley cropping system were followed for a period of 1.5 years (1 maize-cowpea rotation followed by 1 maize season) to a depth of 90 cm, after the application of 15N labeled urea. The highest maize root biomass was found in the 0–10 cm layer and this biomass peaked at 38 and 67 days after planting the 1994 maize (DAP) between the maize rows (112 kg ha−1, on average) and at 38, 67 and 107 DAP under the maize plants (4101 kg ha−1, on average). Almost no maize roots were found below 60 cm at any sampling date. Senna root biomass decreased with time in all soil layers (from 512 to 68 kg ha−1 for the 0–10 cm layer between 0 and 480 DAP). Below 10 cm, at least 62% of the total root biomass consisted of Senna roots and this value increased to 87% between 60 and 90 cm. Although these observations support the existence of a Senna root `safety net' between the alleys which could reduce nutrient leaching losses, the depth of such a net may be limited as the root biomass of the Senna trees in the 60–90 cm layer was below 100 kg ha−1, equivalent to a root length density of only < 0.05 cm cm−3. The proportion of maize root N derived from the applied urea (%Ndfu) decreased significantly with time (from 21% at 21 DAP to 8% at 107 DAP), while %Ndfu of the maize roots at the second harvest (480 DAP) was only 0.6%. The %Ndfu of the Senna roots never exceeded 4% at any depth or sampling time, but decreased less rapidly compared to the %Ndfu of the maize roots. The higher %Ndfu of the maize roots indicates that maize is more efficient in retrieving urea-derived N. The differences in dynamics of the %Ndfu also indicate that the turnover of N through the maize roots is much faster than the turnover of N through the Senna roots. The recovery of applied urea-N by the maize roots was highest in the top 0–10 cm of soil and never exceeded 0.4% (at 38 DAP) between the rows and 7.1% (at 67 DAP) under the rows. Total urea N recovery by the maize roots increased from 1.8 to 3.2% during the 1994 maize season, while the Senna roots never recovered more than 0.8% of the applied urea-N at any time during the experimental period. These values are low and signify that the roots of both plants will only marginally affect the total recovery of the applied urea-N. Measurement of the dynamics of the biomass and N content of the maize and Senna roots helps to explain the observed recovery of applied urea-N in the aboveground compartments of the alley cropping system.

On the enrichment of low-abundant isotopes of light chemical elements by gas centrifuges

A brief review of the main areas for the application of the isotopes 15N and 13C is made. Separation of the nitrogen isotopes in a single gas centrifuge in the form of pure nitrogen, ammonia, and trifluoride of nitrogen as well as the carbon isotopes in the form of carbon dioxide has been studied by means of numerical simulation. The parameters of the centrifugal machine investigated were close to the parameters of the Iguassu machine. The dependence of the efficiency criterion versus the basic parameters of the separation process has been explored in the computational experiments. Comparisons of the calculated results with the experimental data have shown good agreement. The results obtained have demonstrated the possibility of using gas centrifuge technology to enrich successfully the low-abundant isotopes of light chemical elements.

A comparison of different 15N application techniques to study the N net rhizodeposition in the plant-soil system

A comparison of different 15N application techniques to study the N net rhizodeposition in the plant-soil system

Comparison of 15N labelling methods to measure gross nitrogen mineralisation

A laboratory study was conducted to compare methods of labelling the soil NH 4 + pool with 15N as a prerequisite to the measurement of gross N mineralisation. Composite soil (0–12 cm) was collected from 12 sites differing in land use and soil texture. Three methods were used to apply 15N to the soil: (i) addition of a 15NH 3–air gas mixture to the headspace above the soil (gas jar), (ii) injection of a 15NH 3–air gas mixture into re-packed soil cores (gas injector) and (iii) addition of a ( 15NH 4) 2SO 4 solution pipetted onto the soil surface (solution). 15N isotopic dilution was determined between 24 and 72 h after 15N application. Of the applied 15N 52 to 93% was recovered as inorganic N 24 h after application. Less 15N was recovered in the clay loam compared to the sandy loam and silty clay loam for all methods. Recovery from the gas injector was lower than from the gas jar or solution treatments in the sandy loam and silty clay loam. No difference in the recovery of applied 15N between methods was observed in the clay loam. 15N isotopic dilution was less in the gas jar compared to the gas injector and solution treatments. This resulted in significantly lower estimates of gross N mineralisation rates and NH 4 + consumption for the gas jar treatment. The non-uniform distribution of 15N through the soil probably caused these differences as a consequence of 15NH 3 dissolving quickly in the surface of the soil. In seven of the 12 soils, gross rates of N mineralisation were not significantly different when 15N was applied as solution or by gas injection. In the remaining five soils, estimates of gross N mineralisation rates were greater in the solution treatment. Ammonium consumption was significantly different between the solution and gas injector treatments in three soils. Where there were differences in values for gross N mineralisation and NH 4 + consumption rates between methods, the effects were not associated with a particular soil texture or land use. Greater nitrification of 15NH 4 + to 15NO 3 − and shorter pseudo-residence times of the NH 4 + pool were observed in the solution treatment compared to gas injector. This suggests a difference in the distribution of 15N within soil microsites, with the 15N applied in the solution treatment being more accessible for microbial consumption. Nevertheless, application of 15N as an NH 4 + solution, or injection of 15NH 3 gas into soil, resulted in estimates of a similar magnitude for gross N mineralisation rates and identified the same differences between soil type and land use.

SOIL DETRITAL PROCESSES CONTROLLING THE MOVEMENT OF15N TRACERS TO FOREST VEGETATION

Controlled field experiments to study the effects of heightened atmospheric inputs of nitrogen (N) to forests typically demonstrate that most N enters nonextractable pools in soil, while some N is taken up by vegetation, and varying amounts are exported. In a few experimental manipulations of N inputs to forests, 15N has been added as a tracer to more closely study the fates and redistributions of NH4+ and NO3− at the ecosystem level. We developed TRACE, a biogeochemical process model based on previous models, to interpret ecosystem-level 15N field data following applications of 15N-enriched NO3− or NH4+ at the Harvard Forest, Massachusetts, USA. We simulated the forms, masses, atom%, and timing of 15N applications in ambient and chronically fertilized plots over two growing seasons in coniferous and deciduous forest stands. Incorporating principles of stable-isotope redistributions, such as mass balance and pool dilution, into the process model provided a strong means of comparing alternative model formulations against field data. TRACE explicitly illustrated the manner in which rates of gross N turnover in soils could be high enough to provide strong sinks for 15N in ambient plots, while limited enough to allow much greater uptake of 15N by vegetation in fertilized plots. Ectorganic horizons, including litter and humified matter, were key in retaining 15N inputs. We found that fine root uptake and turnover could not account for the rapid movement of 15N into soil pools; direct assimilation into soil pools was required for both NH4+ and NO3− in both deciduous and coniferous forests. Such high rates of N assimilation could not be accounted for by microbial biomass production using detrital C as the substrate. These findings have far-ranging implications for understanding the reciprocal effects of N deposition on forest C budgets, and forest C cycling on ecosystem N retention.

Growth response to fertilisation and recovery of 15N-labelled fertiliser by young hoop pine plantations of subtropical Australia

The stand growth responses to fertilisation were investigated in 1-year-old and 5-year- old second rotation (2R) hoop pine (Araucaria cunninghamii) plantations in subtropical Australia. At the 1-year-old plantation, 4 rates of nitrogen (N) fertiliser (0, 20, 60 and 120 kg N ha-1) were banded either with or without basal fertilisers (BF) containing 60 kg P ha-1 and 50 kg K ha-1. In the 5-year-old plantation, 4 rates of N fertiliser (0, 100, 300, 600 kg N ha-1) were banded with or without the BF. At both sites N fertiliser was applied as ammonium sulphate in 4 equal split dressings over 2 growing seasons. 15N-labelled ammonium sulphate was also used at the 5-year-old plantation in conjunction with the 100 kg N ha-1 treatment. The 15N-labelled fertiliser was applied to 3 trees during the first split application in spring, and to 3 more trees during the second split application in midsummer. The 15N-labelled trees were harvested and the surrounding soil excavated 6 months after the second 15N application. In the 1-year-old plantation, no stand growth response to fertilisation was noted, indicating that fertilisation at plantation establishment was unlikely to increase plantation productivity. In contrast, a significant stand response to N fertilisation was demonstrated at the 5-year-old site. There were no significant differences between the plus N treatments and the increase in basal area and volume due to N fertilisation 2.3 years after commencement of the trial was 45.1 and 43.5%, respectively. No response to basal fertiliser was observed. The mean 15N recovery from the soil–plant system was 79%, with no significant difference noted between the two split applications. The mean 15N recovery in the tree biomass was 56%, with the majority being partitioned into the foliage. On average 21% of the applied 15N could not be accounted for, and was assumed to be lost from the soil–plant ecosystem.

Relationship between rate of crop growth at date of fertiliser N application and fate of fertiliser N applied to winter wheat

To investigate the relationship between the timing of fertiliser N applications and the N use efficiency of wheat, three field experiments with 15N were set up on winter wheat, on three different soils in France. Different crop N demands on the day of fertiliser application were obtained by varying either crop densities or date of fertiliser application. Labelled 15NH415NO3 was applied at tillering and during stem elongation. The 15N recovered from plant and soil at different dates after 15N addition and at maturity of wheat was measured. The fate of fertiliser N was rapidly determined, most of the fertiliser N accumulated in the wheat at maturity having been taken up within a few days of application. 15N recovery by the crop at final harvest (%) varied greatly (19–55% N applied) according to crop density, soil type and date of application. It was linearly related to the instantaneous crop growth rate calculated at the day of 15N application. The amount of fertiliser N immobilised in the soil was constant at 20 kg N ha−1, for all soil types and crop densities. Because residual mineral 15N in the soil at harvest was negligible and immobilisation was constant, the level of total 15N measured in the different N pools (soil+plant) reflected the% 15N uptake by the plant. There was consequently a negative linear relationship between the percentage of 15N not recovered for measurement, and crop growth rate (i.e. crop N demand) at date of fertiliser application. These results suggest that crop N demand at the time of N application determines the ability of the crop to compete for N with other processes, and may be a major factor determining the division of N between soil and crop.

Effects of root-zone temperature on nitrogen accumulation by non-bearing apple trees

SummaryNon-bearing, potted, apple plants were subjected to root temperature of 8.6.1(LRT) or 23.6.18C (HRT) and irrigated with 100 ml of water containing 20 mg of N as NH4NO3, in which both ammonium and nitrate-N fractions were enriched in 15N (10 atom%). The root system of each plant was pressurized (325 kPa) at day 1, 2, 4, and 8 after15 N application to evaluate the transport of nitrogen derived from fertilizer (NDFF) through the xylem. This technique was used to investigate N absorption. LRT reduced the rate of N uptake the day after 15N application relative to HRT. Two, 4 and 8.d after fertilization, the rate of exudation and consequently the uptake rate was similar for LRT and HRT. The total amount of N removed by plants after 8.d was not affected by root treatments. Carbon dioxide assimilation, transpiration rate and stomatal conductance were lower for trees subjected to LRT during the time course of the experiment. We suggest that the delay in N absorption was related mostly to the lower activity of root nitrate reductase detected at the lower root temperature, which increased the nitrate xylem sap concentration and diminished the rate of N uptake.

Potential to improve transfer of N in intercropped systems by optimising host-endophyte combinations

Possibilities for improving N transfer from N2-fixing plants to non-N2-fixing plants by mycorrhiza have been investigated. Initially, the genetic variability with respect to N uptake was assessed by screening five varieties of chicory (Cichorium intybus L.), four of peas (Pisum sativum L.) and three of red clover (Trifolium pratense L.) in combination with eight isolates of arbuscular mycorrhizal fungi. The most promising plant - fungi combinations identified through the cultivar screening were used to optimise conditions for N transfer between intercropped N2-fixing plants (peas and clover) and non-N2-fixing chicory. In the first experiment, the recovery of fixed legume N was investigated using three cultivars, of chicory intercropped with pea variety, and inoculated with one of four mycorrhizal isolates. Roots of the N2-fixing pea and the non-N2-fixing chicory were separated by a root-free soil layer in a three-compartment container. A section of the legume roots was forced to grow into a separate compartment which received four split applications of 15N. The percentage of N in the chicory derived from transfer ranged between 3% and 50%. In a second experiment one chicory variety was intercropped with one red clover variety and inoculated with four mycorrhizal isolates respecetively. A harvest regime was chosen in which the shoots were harvested from intercropped plants at 3,4.5 and 6 months of age. At three months the percentage of N in the chicory derived from transfer ranged between 15% and 18% and at a plant age of 4.5 months from 46 to 77%. At six months the percentage of N in the chicory roots derived from transfer of legume N ranged from 20 to 34% and varied with fungal isolate. Our results show that there is potential for improving N transfer in intercropped plant systems through the methodological selection of suitable plant and mycorrhizal partners.

Turnover of carbon and nitrogen in coniferous forest soils of different N-status and under different 15NH 4N application rate

The aim of the study was to compare the allocation of applied 15NH 4N into soil microbial biomass and soil organic matter (SOM) in mor humus samples from a N-poor and a N-rich plot in an experimental Scots pine ( Pinus sylvestris L.) forest to clarify the N immobilisation mechanisms. Intact humus cores were incubated for 147 days in the laboratory after addition of 4, 20 and 100 μg N g −1 OM of 15N-labelled (NH 4) 2SO 4. CO 2C evolution, extractable organic C, microbial C, labelled and unlabelled NH 4N, NO 3N, extractable organic N, microbial N, and N in soil organic matter (SOM) were determined. Initially, 15NH 4N was rapidly immobilised in all treatments. Microbial assimilation of 15N increased with increased 15N application rate in both the N-poor and N-rich substrate, indicating that NH 4N availability was limiting. No substrate effect was found. Recovery of 15N in microbial biomass ranged from 0.6 to 25% of added 15N and was characterised by an increase in 15N during the first 14 days and a gradual decrease thereafter for nearly all treatments. The recovery of 15N in microbial biomass and SOM showed reciprocal trends after day 14, suggesting a considerable exchange of N between these pools. At the end of the incubation, 36–74% of added 15N occurred in SOM, 2.4–25% was recovered as 15NH 4N, but almost no 15NO 3N was found, indicating no net nitrification. CO 2C evolution was almost constant during the incubation period and was not affected by substrate differences or 15N application rate. Microbial biomass C was highest in the N-poor substrate and was not affected by the 15N application rate. In conclusion, the study showed that lowering the C:N-ratios from 37 to 24 did not significantly alter the turnover of C and N. The retention of 15N in soil microbial biomass and SOM were similar in both substrates when an equal amount of N was added. Microbial N assimilation followed by incorporation of N in SOM were the main N immobilising mechanisms, both in the N-poor and N-rich humus layers.

Absorption of nitrogen by Japanese mint from mineralization and immobilization by the soil microbial biomass

Laboratory incubation and greenhouse experiments were conducted with two soils having contrasting physico‐chemical characteristics to evaluate nitrogen (N) mineralization, immobilization in soil microbial biomass, and accumulation in Japanese mint (Mentha arvensis L.) using labeled (15NH4)2SO4, applied at 0, 50, and 100 mg#lbkg‐1 soil. Rate of mineralization in soils varied from 0.08 to 2.21 μg#lbg‐1#lbday‐1. Fertilizer application increased the mineralization of native soil N. About 22 to 60% of the applied 15N was recovered in the soil microbial biomass during the growth period of mint (January‐June). Relative contribution of fertilizer 15N towards total N uptake by mint at maturity was 42–54% in soil I and 35 to 55% in soil II. Contribution of soil N towards total N accumulation increased with the doses of 15N application.

Temporary Biological N Cońservation by Winter Oilseed Turnip (Brassica Rapa L.) and its Influence on Following Crops and N-Percolation

As shown in field plot trials with application of 15N on loamy sand (albic luvisol), winter oilseed turnip (Brassica rapa L.; ssp. oleifera (Dc.) METZG.) incorporated considerable N amounts during the winter period. By this a reduction of N percolation into deeper soil layers was achieved. After mulching, about 30% of the catch crop N was taken up by the following crop, maize (Zea mays L., cv. ‘BEKENOVA’). Maize covered 11% of its total N demand deriving from the catch crop.

Lysimeter Investigations on the Effect of Winter Catch Crops and Weeded Fallow on the N-Dynamics in a Sandy Treposol Soil of Northeast Germany

Lysimeter experiments (soil: sandy treposol, from the region “Havelländisches Luch”, Brandenburg, Germany) with application of 15N labelled fertilizer (80 kg N per ha as 15NH4 15NO3, 10 at.-%15N exc.; for simulating mineralization in the early autumn period) were carried out to determine to what extent the amount of mineral- N was temporary conserved by winter catch crops, taken up subsequently in the vegetation periods by following crops, taken by subsequently in the vegetation periods by following crops, or percolated in the leaching water, respectively. The results were as follows: 1) Until winter or spring respectively, the catch crop uptake rates of applied mineral-N were 32% for phacelia (Phacelia tanacetifolia BENTH.), 25% for winter rape (Brassica napus L. cv. ‘AKELA’), and 16% for white mustard (Sinapis alba L.). 2) In the year after, following maize incorporated from 2.1 to 4.5% of the fertilizerborne N. The following plant community of fallow took up from 0.2 to 0.5% N originating from the fertilizer-N. 3) In comparison with the catch crops, N-leaching losses under fallow conditions were highest and equivalent to 17% of the applied fertilizer-N amount. In contrast to 3% of white mustard, phacelia and winter rape reduced N-leaching losses to 0.2 and 0.3% of the applied fertilizer-N amount. 4) In spring of the first year after the beginning of investigations, N-leaching losses were highest under fallow conditions and white mustard cultivation. Thus, the amounts of nitrate losses would exceed the EU limit for drinking water. 5) Three years after the investigations had been started, 10% (white mustard) and 20% (fallow) of the applied fertilizer-N was still found in th lysimeter soil.

Apparent Atmospheric Nitrogen Loss from Hydroponically Grown Corn

AbstractIsotopic 15N tracers have been important tools in the understanding of N cycling in agricultural and natural soil‐plant systems. The objectives of this research were to quantify losses of 15N under controlled conditions, evaluate 15NH3 transport between plants, and assess the potential for error associated with the use of N isotopes. Corn (Zea mays L.) was grown hydroponically in 5‐ to 15‐mm‐diam. gravel in twelve 162‐L barrels. All N was applied as NO3 with enrichment periods of full season, planting through anthesis, anthesis to harvest, or no 15N applied. Plant growth and total N accumulation followed normal growth curves and maximum N content was obtained at anthesis. When 15N treatments stopped at anthesis, plants lost 15N (presumably as 15NH3) equivalent to about 23% of total plant N between anthesis and harvest. In treatments in which 15N applications started at anthesis, total N content in the leaves and stems decreased between anthesis and harvest while 15N content increased indicating a substitution of recently absorbed N for N compounds translocated to the fruit. Volatile losses of N indicate that caution must be exercised when conducting and interpreting 15N experiments. Significant plant‐to‐plant transport of 15NH3 showed that decreases in plant 15N content could not be used to estimate NH3 volatilization for an entire field.

Specific labeling approaches to guanine and adenine imino and amino proton assignments in the AMP-RNA aptamer complex.

The secondary structure of a recently identified ATP-binding RNA aptamer consists of a purine-rich 11-residue internal loop positioned opposite a single guanine bulge flanked on either side by helical stem segments. The ATP ligand targets the internal loop and bulge domains, inducing a structural transition in this RNA segment on complex formation. Specifically, 10 new slowly exchanging proton resonances in the imino, amino and sugar hydroxyl chemical shift range are observed on AMP-RNA aptamer complex formation. This paper outlines site-specific labeling approaches to identify slowly exchanging imino (guanine) and amino (guanine and adenine) protons in internal loop and bulge segments of compact RNA folds such as found in the AMP-RNA aptamer complex. One approach incorporates 15N-labeled guanine (N1 imino and N2 amino positions) and 15N-labeled adenine (N6 amino position), one residue at a time, in the AMP-binding RNA aptamer, with labeling incorporation through chemical synthesis facilitated by generating the aptamer from two separate strands. The unambiguous assignments deduced from the 15N labeling studies have been verified from an independent labeling strategy where individual guanines in the internal loop have been replaced, one at a time, by inosines and assignments were made on the basis of the large 2 ppm downfield shift of the guanine imino protons on inosine substitution. The strengths and limitations of the inosine-for-guanine substitution approach emerge from our studies on the AMP-RNA aptamer complex. The assignment of the internal loop and bulge imino and amino protons was critical in our efforts to define the solution structure of the AMP-RNA aptamer complex since these slowly exchanging protons exhibit a large number of long-range intramolecular NOEs within the RNA, as well as intermolecular NOEs to the AMP in the complex. The current application of specific 15N and inosine labeling approaches for exchangeable imino and imino proton assignments in the nonhelical segments of an RNA aptamer complex in our laboratory complements selective 2H and 13C approaches to assign nonexchangeable base and sugar protons in RNA and ligand-RNA complexes reported in the literature.

Field measurements of nitrogen fixation in leguminous trees used in agroforestry systems: Influence of 15N-labeling approaches and reference trees

Appropriate 15N-labeling methods are crucial for estimating N2-fixation in trees used in agroforestry systems. A 4-year field experiment was conducted on an Alfisol in Southwestern Nigeria to compare the estimates of N2 fixed in Leucaena leucocephala, using two non-N2-fixing leguminous trees, Senna siamea and S. spectabilis, as reference plants and three different methods of introducing 15N into soil. The atom % 15N uptake pattern (as reflected in the leaves) was identical in both N2- and non-N2-fixing tree species irrespective of the 15N-application method. There was a significant decline in atom % 15N excess in the leaves of L. leucocephala (from 0.266 to 0.039), S. siamea (0.625 to 0.121), and S. spectabilis (from 0.683 to 0.118) from the first sampling 12 months after planting and the second sampling 18 months after sampling. From the second harvest in 1991 until the end of the experiment (fifth) harvest in 1993, however, the atom 15N % excess decline in leaves of the three species was less pronounced and depended on the method of 15N application. In those plants to which the tracer was applied once at planting, the 15N decline was steady between the second and the last prunings. In the split-application treatment, the atom 15N % excess increased slightly at the third pruning and decreased during the subsequent two prunings. The reference tree and the method of 15N application influenced the estimated proportion of N derived from atmospheric N2 by L. leucocephala, calculated as 73 and 64%, corresponding to 119 and 98 kg N ha-1 of N2 fixed per 6 months, when S. spectabilis and S. siamea were used as reference trees, respectively. The approach by which 15N-labeled fertilizer was applied to the soil in three splits gave slightly higher estimates of N derived from the atmosphere but this was of little agronomic significance because total N2 fixed was similar for all methods.

Application of 15N and xylem ureide methods for assessing N2 fixation of three shrub legumes periodically pruned for forage

The apparently diminished capacity for N2 fixation by the shrub legume Calliandra calothyrsus (Calliandra) relative to other woody perennial legumes was investigated in a field experiment in northern Queensland, Australia. In this trial, (i) the proportion of plant nitrogen (N) derived from symbiotic N2 fixation (%Pfix) and the amounts of N2 fixed were compared in Calliandra, Gliricidia sepium (Gliricidia) and Codariocalyx gyroides (Codariocalyx), (ii) variations in N2 fixation due to season or tree age were determined, (iii) estimates of Pfix derived with the 15N natural abundance technique were compared with values obtained from 15N enrichment or xylem sap ureide procedures to determine whether the previous conclusions about Calliandra's ability to fix N had resulted from specific problems with the natural abundance methodology used in the earlier studies.

How Nitrogen Supply Affects Growth and Nitrogen Uptake, Use Efficiency, and Loss from Citrus Seedlings

We examined how N supply affected plant growth and N uptake, allocation and leaching losses from a fine sandy soil with four Citrus rootstock species. Seedlings of `Cleopatra' mandarin (Citrus reticulata Blanco) and `Swingle' citrumelo (C. paradisi × P. trifoliata) were grown in a glasshouse in 2.3-liter pots of Candler fine sand and fertilized weekly with a complete nutrient solution containing 200 mg N/liter (20 mg N/week). A single application of 15NH415NO3(17.8% atom excess 15N) was substituted for a normal weekly N application when the seedlings were 22 weeks old (day O). Six replicate plants of each species were harvested at 0.5, 1.5, 3.5, 7, 11, and 30 days after 15N application. In a second experiment, NH4 NO3 was supplied at 18,53, and 105 mg N/week to 14-week-old `Volkamer' lemon (C. volkameriana Ten. &amp; Pasq.) and sour orange (C. aurantium L.) seedlings in a complete nutrient solution for 8 weeks. A single application of 15NH415NO3 (23.0% 15N) was substituted at 22 weeks (day 0), as in the first experiment, and seedlings harvested 3,7, and 31 days after 15N application. Nitrogen uptake and partitioning were similar among species within each rate, but were strongly influenced by total N supply and the N demand by new growth. There was no 15N retranslocation to new tissue at the highest (105 mg N/week) rate, but N supplies below this rate limited plant growth without short-term 15N reallocation from other tissues. Leaf N concentration increased linearly with N supply up to the highest rate, while leaf chlorophyll concentration did not increase above that at 53 mg N/week. Photosynthetic CO2 assimilation was not limited by N in this study; leaf N concentration exceeded 100 mmol·m-2 in all treatments. Thus, differences in net productivity at the higher N rates appeared to be a function of increased leaf area, but not of leaf N concentration. Hence, N use efficiency decreased significantly over the range of N supply, whether expressed either on a gas-exchange or dry weight basis. Mean plant 15N uptake efficiencies after 31 days decreased from 60% to 47% of the 15N applied at the 18,20, and 53 mg N/week rates to less than 33% at the 105 mg N/week rate. Leaching losses increased with N rate, with plant growth rates and the subsequent N requirements of these Citrus species interacting with residual soil N and potential leaching loss.