Perspectives on measurement of denitrification in the field including recommended protocols for acetylene based methods

Of the biogeochemical processes, denitrification has perhaps been the most difficult to study in the field because of the inability to measure the product of the process. The last decade of research, however, has provided both acetylene and¹⁵N based methods as well as undisturbed soil core andin situ soil cover sampling approaches to implementing these methods. All of these methods, if used appropriately, give comparable results. Thus, we now have several methods, each with advantages for particular sites or objectives, that accurately measure denitrification in nature. Because of the general usefulness of the acetylene methods, updated protocols for the following three methods are given: gas-phase recirculation soil cores; static soil cores; and the denitrifying enzyme assay also known as the phase 1 assay. Despite the availability of these and other methods, denitrification budgets remain difficult to accurately establish in most environments because of the high spatial and temporal variability inherent in denitrification. Appropriate analysis of those data includes a distribution analysis of the data, and if highly skewed as is typically the case, the most accurate method to estimate the mean and the population variance is the UMVUE method (uniformly minimum variance unbiased estimator). Geostatistical methods have also been employed to improve spatial and temporal estimates of denitrification. These have occasionally been successful for spatial analysis but in the attempt described here for temporal analysis the approach was not useful.Discussions of the importance of denitrification have always focused on quantifying the process and whether particular measured quantities are judged to be a significant amount of nitrogen. A second line of evidence discussed here is the extant genetic record that results from natural selection. These analysis lead to the conclusion that strong selection for denitrification must currently be occurring, which implies that the process is of general significance in soils.     

Modeling long-term crop response to fertilizer and soil nitrogen

A simple nitrogen balance model to calculate long-term changes in soil organic nitrogen, nitrogen uptake by the crop and recovery of applied nitrogen, is presented. It functions with time intervals of one year or one growing season. In the model a labile and a stable pool of soil organic nitrogen are distinguished. Transfer coefficients for the various inputs of nitrogen are established that specify the fractions taken up by the crop, lost from the system, and incorporated in soil organic nitrogen. It is shown how input data, model parameters and initial pool sizes can be derived and how the model can be used for calculating long-term changes in total soil organic nitrogen and uptake by the crop. For nitrogen applied annually as fertilizer or organic material the time course of nitrogen uptake and recovery of applied nitrogen is calculated. To test the sensitivity of the model, calculations have been performed for different environmental conditions with higher or lower risks for losses. The model has also been applied to establish fertilizer recommendations for a certain target nitrogen uptake by the crop. Finally, for agricultural systems where periods of cropping alternate with peroids of green fallow the time course of nitrogen uptake by the crop is calculated.     

Consequences of terbium (111) binding on the conformation and enzymatic activity of Guinea pig liver transglutaminase

Calcium ions are crucial for expression of transglutaminase activity. Although lanthanides have been reported to substitute for calcium in a variety of protein functions, they did not replace the calcium requirement during transglutaminase activity measurements. Furthermore, lanthanides strongly inhibited purified liver transglutaminase activity using either casein or fibrinogen as substrates. Terbium (III) inhibition of transglutaminase-catalyzed putrescine incorporation into casein was not reversed by the presence of 10–200 fold molar excess of calcium ions (Ki for Tb(III)=60 µM). Conformational changes in purified liver transglutaminase upon Tb(III) binding were evident from a biphasic effect of Tb(III) on transglutaminase binding to fibrin. Low concentrations of Tb(III) (1 µM to 10 µM inhibited the binding of transglutaminase to fibrin, whereas higher concentrations (20 µM to 100 µM promoted binding. Conformational changes in purified liver transglutaminase consequent to Tb(III) binding were also demonstrated by fluorescence spectroscopy due to Forster energy transfer. Fluorescence emission was stable to the presence of 200 mM NaCl and 100 mM CaCl₂ only partially quenched emission. Purified liver transglutaminase strongly bound to Tb(III)-Chelating Sepharose beads and binding could not be disrupted by 100 mM CaCl₂ solution. Our data suggest that Tb(III)-induced conformational changes in transglutaminase are responsible for the observed effects on enzyme structure and function. The potential applications of Tb(III)-transglutaminase interactions in elucidating the structure-function relationships of liver transglutaminase are discussed.     

Limited chloroplast gene transfer via recombination overcomes plastomegenome incompatibility between Nicotiana tabacum and Solanum tuberosum

Green cybrids with a new nucleus-chloroplast combination cannot be selected after protoplast fusion in the intersubfamilial Nicotiana-Solanum combination. As an approach to overcome the supposed plastomegenome incompatibility, a partial plastome transfer by genetic recombination has been considered. After fusions of protoplasts of a light-sensitive Nicotiana tabacum (tobacco) plastome mutant and lethally irradiated protoplasts of wild-type Solanum tuberosum (potato), a single green colony was recovered among 2.5×10⁴ colonies. The regenerated plants had tobacco-like (although abnormal) morphology, but were normally green, and sensitive to tentoxin, demonstrating chloroplast markers of the potato parent. Restriction enzyme analysis of the chloroplast DNA (cpDNA) revealed recombinant, nonparental patterns. A comparison with physical maps of the parental cpDNA demonstrated the presence of a considerable part of the potato plastome flanked by tobacco-specific regions. This potacco plastome proved to be stable in backcross and backfusion experiments, and normally functional in the presence solely of N. tabacum nucleus.     

An empirical analysis of the factors contributing to 20-year decrease in soil pH in an old-field plantation of loblolly pine

The pH of weak-acid solutions is controlled by acid concentration (HA + A^–), the degree of acid dissociation (A^–/HA), and the strength of the acids present (pK_a). We developed an empirical approach that allows the relative importance of each of these factors to be estimated for soils. This empirical model was applied to soils collected from an old-field plantation of loblolly pine (Pinus taeda L.) at 5 and 25 years of age. During this period, soil pH dropped by 0.3 to 0.8 units, and extractable calcium, magnesium and potassium declined by 20 to 80%. The empirical model indicates that the decline in pH resulted largely from the reduction in base saturation of the exchange complex. However, the average acid strength of the exchange complex decreased during the 20 years, preventing a greater decline of perhaps 0.1 to 0.2 units in the observed pH. The rate of decrease in the acid neutralizing capacity to pH 3.5 was about 1.3 kmol_c/ha annually, while the increase in base neutralizing capacity was about 2.7 and 1.6 kmol_c/ha annually to pH 5.5 and 8.2, respectively. Extractable alkali and alkaline earth cations declined by about 2.2 kmol_c/ha annually, matched by the rate of increase in aluminium. These changes demonstrated the dynamic nature of poorly buffered soils, and indicated that changes in soil acidity may be expected over a period of decades (especially following changes in land-use).     

Soil N mineralization and nitrification in relation to nitrogen solution chemistry in a small forested watershed

Spatial variations in soil processes regulating mineral N losses to streams were studied in a small watershed near Toronto, Ontario. Annual net N mineralization in the 0–8 cm soil was measured in adjacent upland and riparian forest stands using in situ soil incubations from April 1985 to 1987. Mean annual rates of soil N mineralization and nitrification were higher in a maple soil (93.8 and 87.0 kg.ha^–1) than in a pine soil (23.3 and 8.2 kg.ha^–1 ). Very low mean rates of mineralization (3.3 kg.ha^–1) and nitrification (3.4 kg.ha^–1) were found in a riparian hemlock stand. Average NO₃-N concentrations in soil solutions were 0.3–1.0 mg.L^–1 in the maple stand and >0.06mg.L^–1 in the pine stand. Concentrations of NO₃–N in shallow ground water and stream water were 3–4× greater in a maple subwatershed than in a pine subwatershed. Rapid N uptake by vegetation was an important mechanism reducing solution losses of NO₃–N in the maple stand. Low rates of nitrification were mainly responsible for negligible NO₃–N solution losses in the pine stand.     

Hydro-sedimentology of the Johnstone River estuary

This paper examines the physical consequences of increased catchment sediment yields on the sediment budget and the hydrodynamic setting of the South Johnstone River estuary in North Queensland. A combined study involving hydrological monitoring, assessment of sediment sources, estimation of riverine sediment budget and hydro-sedimentological numerical modelling for estuarine sediment transport is currently underway. Initial field and laboratory observations indicate that the sediment delivery from highly erosion-prone sugar cane cultivations in the tropical catchment has increased dramatically during the last 10 years. This has subsequently given rise to elevated flood levels in both the lower and upper catchment areas, as well as significant modifications to the river bed morphology.     

Sensitive bioassays for determining residues of sulfonylurea herbicides in soil and their availability to crop plants

Sulfonylurea herbicides are potent inhibitors of plant growth and are extremely active against a wide spectrum of weeds. They are used at very low rates (10–50 g ai/ha) and cause rapid inhibition of root and shoot growth of young plants. Routine chemical assays for detecting low levels of these compounds are difficult and there is need to develop sensitive bioassay methods for detecting their extremely low residue levels in the soil.This paper describes a simple pot bioassay method with a self watering system using turnip (Brassica rapa) seedlings as test plants for quantitative determination of sulfonylurea herbicides. Results are presented with six of these compounds whose activity was investigated in widely differing substrates. The potential availability to plants was calculated from the dose-response curves in different substrates. The dose-response relationship has been described by a specifically developed computer model. Details are also given of a direct seeded bioassay method with controlled watering system using several test species for detection of sulfonylurea herbicides. The potential uses and practical applications of both techniques are discussed.     

Preparation of polysaccharide-enzyme conjugates for competitive binding assays

A variety of bacterial O-polysaccharides were covalently linked to enzymes and it was demonstrated with three discrete monoclonal antibodies that enzyme-glycoconjugates function as convenient labelled antigens in direct enzyme immunoassays, particularly competitive assays that quantify bacterial O-antigens. Two strategies, each involving reductive amination, were used to couple O-polysaccharides to enzymes, while retaining high enzymic activity. Reduction of the Schiff base formed between, 1,3-diaminopropane and the terminal reducing ketodeoxyoctanoic acid (KDO) residue present in the majority of the lipopolysaccharide (LPS) core domains, following mild acid removal of Lipid A, offered the most direct route to mono-aminated polysaccharide. Alternatively, mild periodate oxidation of KDO and heptose residues generated multiple aldehyde targets for Schiff base formation, without affecting the O-antigenic determinant. Hetero- and homobifunctional coupling reagents, sulphosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate and discuccinimidyl suberate, activated polysaccharide for coupling to enzymes at amino and sulphydryl sites and produced conjugates that retained at least 95% of the original enzymic activity. The most suitable enzyme conjugates, especially for competitive inhibition EIA were those bearing one polysaccharide chain, and these were easily prepared from horse-radish peroxidase. Although the extent of conjugation of activated polysaccharide to -galactosidase and alkaline phosphatase could be controlled by reaction stoichiometry, the use of these enzymes was a less effective utilization of valuable antigen and enzyme.Issued as NRCC No. 31634