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.     

Cycling of nutrients from dying roots to living plants,including the role of mycorrhizas

This paper presents information about the release of nitrogen and phosphorus from dying grass roots and the capture of phosphorus by other, living plants. We have paid particular attention to the part played by mycorrhizas in this phosphorus capture, and the possible importance of mycorrhizal links between dying and living roots.WhenLolium perenne plants were grown with ample nutrients and their roots then detached and buried in soil, about half the nitrogen and two-thirds of the phosphorus was lost in three weeks, but only one-fifth of the dry weight. The C:N and C:P ratios suggest that microbial growth in the roots would at first be C-limited but would become N- and P-limited within three weeks.Rapid transfer of³²P can occur from dying roots to those of a living plant if the two root systems are intermingled. The amount transferred was substantially increased in two species-combinations that are known to form mycorrhizal links between their root systems. In contrast, in a species-combination where only the living (receiver) plant could become mycorrhizal no significant increase of³²P transfer occurred. This evidence, although far from conclusive, suggests that mycorrhizal links between dying and living roots can contribute to nutrient cycling. This research indicates a major difference in nutrient cycling processes between perennial and annual crops.     

Sulphate influx in wheat and barley roots becomes more sensitive to specific protein-binding reagents when plants are sulphate-deficient

When young wheat (Triticum aestivum L.) or barley (Hordeum vulgare L.) plants were deprived of an external sulphate supply (-S plants), the capacity of their roots to absorb sulphate, but not phosphate or potassium, increased rapidly (derepression) so that after 3–5 d it was more than tenfold that of sulphate-sufficient plants (+S plants). This increased capacity was lost rapidly (repression) over a 24-h period when the sulphate supply was restored. There was little effect on the uptake of L-methionine during de-repression of the sulphate-transport system, but S input from methionine during a 24-h pretreatment repressed sulphate influx in both+S and-S plants.Sulphate influx of both+S and-S plants was inhibited by pretreating roots for 1 h with 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS) at concentrations > 0.1 mol · m^-3. This inhibition was substantially reversed by washing for 1 h in DIDS-free medium before measuring influx. Longer-term pretreatment of roots with 0.1 mol·m^-3 DIDS delayed de-repression of the sulphatetransport system in-S plants but had no influence on+S plants in 3 d.The sulphydryl-binding reagent, n-ethylmaleimide, was a very potent inhibitor of sulphate influx in-S roots, but was much less inhibitory in +S roots. Its effects were essentially irreversible and were proportionately the same at all sulphate concentrations within the range of operation of the high-affinity sulphate-transport system. Inhibition of influx was 85–96% by 300 s pretreatment by 0.3 mol·m^-3 n-ethylmaleimide. No protection of the transport system could be observed by including up to 50 mol·m^-3 sulphate in the n-ethylmaleimide pre-treatment solution. A similar differential sensitivity of-S and+S plants was seen with p-chloromercuriphenyl sulphonic acid.The arginyl-binding reagent, phenylglyoxal, supplied to roots at 0.25 or 1 mol·m^-3 strongly inhibited influx in-S wheat plants (by up to 95%) but reduced influx by only one-half in+S plants. The inhibition of sulphate influx in-S plants was much greater than that of phosphate influx and could not be prevented by relatively high (100 mol·m^-3 sulphate concentrations accompanying phenylglyoxal treatment. Effects of phenylglyoxal pretreatment were unchanged for at least 30 min after its removal from the solution but thereafter the capacity for sulphate influx was restored. The amount of new carrier appearing in-S roots was far greater than in+S roots over a 24-h period.The results indicate that, in the de-repressed state, the sulphate transporter is more sensitive to reagents binding sulphydryl and arginyl residues. This suggests a number of strategies for identifying the proteins involved in sulphate transport.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonic acid - NEM n-ethylmaleimide - PCMBS p-chloromercuriphenyl sulphonic acid     

Charge and acidity compensation during proton-sugar symport in Chlorella: The H+-ATPase does not fully compensate for the sugar-coupled proton influx

This study was undertaken in order to demonstrate the extent to which the activity of the plasmalemma H⁺-ATPase compensates for the charge and acidity flow caused by the sugar-proton symport in cells of chlorella vulgaris Beij.. Detailed analysis of H⁺ and K⁺ fluxes from and into the medium together with measurements of respiration, cytoplasmic pH, and cellular ATP-levels indicate three consecutive phases after the onset of H⁺ symport. Phase 1 occurred immediately after addition of sugar, with an uptake of H⁺ by the hexoseproton symport and charge compensation by K⁺ loss from the cells and, to a smaller degree, by loss of another ion, probably a divalent cation. This phase coincided with strong membrane depolarization. Phase 2 started approximately 5 s after addition of sugar, when the acceleration of the H⁺-ATPase caused a slow-down of the K⁺ efflux, a decrease in the cellular ATP level and an increase in respiration. The increased respiration was most probably responsible for a pronounced net acidification of the medium. This phase was inhibited in deuterium oxide. In phase 3, finally, a slow rate of net H⁺ uptake and K⁺ loss was established for several further minutes, together with a slight depolarization of the membrane. There was hardly any pH change in the cytoplasm, because the cytoplasmic buffering capacity was high enough to stabilize the pH for several minutes despite the net H⁺ fluxes. The quantitative participation of the several phases of H⁺ and K⁺ flow depended on the pH of the medium, the ambient Ca²⁺ concentration, and the metabolic fate of the transported sugar. The results indicate that the activity of the H⁺-ATPase never fully compensated for H⁺ uptake by the sugar-symport system, because at least 10% of symport-caused charge inflow was compensated for by K⁺ efflux. The restoration of pH in the cytoplasm and in the medium was probably achieved by metabolic reactions connected to increased glycolysis and respiration.Abbreviations DMO dimethyloxazolidinedione - EDTA ethylcnediaminetetraacetic acid - p.c. packed cell volume     

Nonthermal effect of microwave irradiation on nitrite uptake in Chlamydomonas reinhardtii

When cells of the unicellular green alga Chlamydomonas reinhardtii were subjected to microwave irradiation at 2.45 GHz, nitrite uptake kinetics still obeyed the Michaelis-Menten equation, the Km of the process remaining constant, whereas V _max increased, which indicates an enhanced nonthermal permeability in irradiated cells.     

Growth and nitrate uptake properties of plants grown at different relative rates of nitrogen supply. II. Activity and affinity of the nitrate uptake system in Pisum and Lemna in relation to nitrogen availability and nitrogen demand

Abstract Net nitrate uptake rates were measured and the kinetics calculated in non-nodulated Pisum sativum L. cv. Marma and Lemna gibba L. adapted to constant relative rates of nitrate-N additions (R_A), ranging from 0.03 to 0.27 d^?1 for Pisum and from 0.05 to 0.40 d^?1 for Lemna, V_max of net nitrate uptake (measured in the range 10 to 100 mmol m^?3 nitrate, i.e. ‘system I’) increased with R_A in the growth limiting range but decreased when R_A exceeded the relative growth rate (RGR), K_m was not significantly related to changes in R_A. On the basis of previous ¹³N-flux experiments, it is concluded that the differences in V_max at growth limiting R_A are attributable to differences in influx rates. Linear relationships between V_max and tissue nitrogen concentrations were obtained in the growth limiting range for both species, and extrapolated intercepts relate well with the previously defined minimal nitrogen concentrations for plant growth (Oscarson, Ingemarsson & Larsson, 1989). Analysis of V_max for net nitrate uptake on intact plant basis in relation to nitrogen demand during stable, nitrogen limited, growth shows an increased overcapacity at lower R_A values in both species, which is largely explained by the increased relative root size at low R_A. A balancing nitrate concentration, defined as the steady state concentration needed to sustain the relative rate of increase in plant nitrogen (R_N), predicted by R_A, was calculated for both species. In the growth limiting range, this value ranges from 3.5 mmol m^?3 (R_A 0.03 d^?1) to 44 mmol m^?3 (R_A 0.21 d^?1) for Pisum and from 0.2 mmol m^?3 (R_A 0.05 d^?1) to 5.4 mmol m^?3 (R_A 0.03 d^?1) for Lemna. It is suggested that this value can be used as a unifying measure of the affinity for nitrate, integrating the performance of the nitrate uptake system with nitrate flux and long term growth and demand for nitrogen.     

On the mechanism of cellular cadmium uptake

Review of the available evidence on the mechanism of cellular Cd uptake in the rat jejunum supports the concept that this process consists of nonspecific binding to anionic sites on the membrane, followed by a temperature-dependent and rate-limiting internalization step. Because temperature-sensitive transmembrane movement of Cd can be demonstrated also in isolated brush-border vesicles and in erythrocyte ghosts, it is not likely to result from pinocytosis but may be related directly to membrane fluidity. There is no need to assume the existence of saturable Cd carriers, or competition of Cd with essential polyvalent cations for their specific transport systems. Uptake of Cd by tubular epithelium in the kidney of the intact rabbit appears to resemble that described for the jejunum, with the internalization step limiting the rate of uptake.     

Development of a liquid flow-through system to inhibit browning in callus cultures of guayule (Parthenium argentatum Gray)

Calli of P. argentatum were grown on a newly designed liquid nutrient flow-through system which facilitated the subculturing of calli and delayed browning for 6 weeks. Friable calli were obtained on half-strength Gamborg B5-medium supplemented with 0.05 mgl⁻¹ 2,4-dichlorophenoxyacetic acid. Shoots developed on media supplemented with 0.2 mgl⁻¹ benzylaminopurine but lacking 2,4-dichlorophenocyacetic acid.     

The biennial life strategy in a random environment

In a previous paper (J. Math. Biol. 26, 199–215 (1988)) we calculated the mean and variance of the long-run geometric growth rate of a discrete-time population model with two age classes in a random environment. The formula which was used in that paper as the starting point for the computation of the variance represents only the contribution of the one-period variances. Here we supplement these results by a calculation of the exact variance. All qualitative conclusions reached before are unaffected.