Composition of the xylem sap of tomato seedlings cultivated on media with HCO3 − and nitrogen source as NO3 − or NH4 +

The results of the experiments discussed here present changes in the chemical composition of xylem sap of tomato seedlings cultivated in hydroponics on media containing 5 mmol HCO₃ ^– and an N-source given as NO₃ ^–, NH₄ ⁺ or these two forms in different proportions. The occurrence of free NH₄ ⁺ in the xylem sap of NH₄ ⁺-seedlings and in NO₃ ^–-seedlings indicates that the process of N-assimilation was not only confined to roots. The application of HCO₃ ^– to the medium effected a decrease in the concentration of NH₄ ⁺ in the xylem sap of NH₄ ⁺-seedlings, having no effect on changes in the concentration of NO₃ ^– or NH₄ ⁺ in NO₃ ^–-seedlings. Malate, citrate, fumarate, and succinate were identified in the xylem sap. The concentration of carboxylates in NO₃ ^–-seedlings exceeded by about 50% that recorded in NH₄ ⁺-seedlings. The highest concentration of malate constituting from 80% to 93.5% of this fraction, was determined in this group of compounds. The enrichment of the medium with HCO₃ ^– ions induced an increase in the content of carboxylates, chiefly of malate. In these experimental conditions an increase in the malate concentration in the xylem sap of NO₃ ^– and NH₄ ⁺-seedlings reached relative values of 100% and 36%, respectively. The total concentration of amides and amino acids was about 2.6 times higher in the xylem sap of NH₄ ⁺-seedlings than in NO₃ ^–-seedlings. Amide glutamine was the main component of this fraction in xylem sap and its total concentration was about 3.3 times higher in NH₄ ⁺-seedlings than that determined in NO₃ ^–-seedlings. Glutamine, glutamate, aspargine, and aspartate constituted from 69% to 77% of this fraction. The concentration of the remaining amino acids varied from 0.6% to 7%. The enrichment of the medium with HCO₃ ^– ions also effected an increase in the concentration of amides and amino acids in the xylem sap by about 17% and 56% in the case of NO₃ ^– and NH₄ ⁺-seedlings, respectively, in comparison with the respective controls (without HCO₃ ^–). Abbreviations: DAG – days after germination; DIC – dissolved inorganic carbon; GOGAT – glutamine:2-oxoglutarate aminotransferase; GS – glutamine synthetase; PAR – photosynthetically active radiation; PEPc – phosphoenolpyruvate carboxylase     

The seasonal dynamics of amino acids and other nutrients in Alaskan Arctic tundra soils

Past research strongly indicates the importance of amino acids in the N economy of the Arctic tundra, but little is known about the seasonal dynamics of amino acids in tundra soils. We repeatedly sampled soils from tussock, shrub, and wet sedge tundra communities in the summers of 2000 and 2001 and extracted them with water (H₂O) and potassium sulfate (K₂SO₄) to determine the seasonal dynamics of soil amino acids, ammonium (NH₄⁺), nitrate (NO₃^–), dissolved organic nitrogen (DON), dissolved organic carbon (DOC), and phosphate (PO₄^2–). In the H₂O extractions mean concentrations of total free amino acids (TFAA) were higher than NH₄⁺ in all soils but shrub. TFAA and NH₄⁺ were highest in wet sedge and tussock soils and lowest in shrub soil. The most predominant amino acids were alanine, arginine, glycine, serine, and threonine. None of the highest amino acids were significantly different than NH₄⁺ in any soil but shrub, in which NH₄⁺ was significantly higher than all of the highest individual amino acids. Mean NO₃^– concentrations were not significantly different from mean TFAA and NH₄⁺ concentrations in any soil but tussock, where NO₃^– was significantly higher than NH₄⁺. In all soils amino acid and NH₄⁺ concentrations dropped to barely detectable levels in the middle of July, suggesting intense competition for N at the height of the growing season. In all soils but tussock, amino acid and NH₄⁺ concentrations rebounded in August as the end of the Arctic growing season approached and plant N demand decreased. This pattern suggests that low N concentrations in tundra soils at the height of the growing season are likely the result of an increase in soil N uptake associated with the peak in plant growth, either directly by roots or indirectly by microbes fueled by increased root C inputs in mid-July. As N availability decreased in July, PO₄^2– concentrations in the K₂SO₄ extractions increased dramatically in all soils but shrub, where there was a comparable increase in PO₄^2– later in the growing season. Previous research suggests that these increases in PO₄^2– concentrations are due to the mineralization of organic phosphorus by phosphatase enzymes associated with soil microbes and plant roots, and that they may have been caused by an increase in organic P availability.     

Growth responses of the perennial legume Sesbania sesban to NH4 and NO3 nutrition and effects on root nodulation

Preference for NH₄⁺ or NO₃⁻ nutrition by the perennial legume Sesbania sesban (L.) Merr. was assessed by supplying plants with NH₄⁺ and NO₃⁻ alone or mixed at equal concentrations (0.5 mM) in hydroponic culture. In addition, growth responses of S. sesban to NH₄⁺ and NO₃⁻ nutrition and the effects on root nodulation and nutrient and mineral composition of the plant tissues were evaluated in a hydroponic setup at a range of external concentration of NH₄⁺ and NO₃⁻ (0, 0.1, 0.2, 0.5, 2 and 5 mM). Seedlings of S. sesban grew equally well when supplied with either NH₄⁺ or NO₃⁻ alone or mixed and had high relative growth rates (RGRs) ranging between 0.19 and 0.21 d⁻¹. When larger plants of S. sesban were supplied with NH₄⁺ or NO₃⁻ alone, the RGRs and shoot elongation rates were not affected by the external concentration of inorganic N. At external N concentrations up to 0.5 mM nodulation occurred and contributed to the N nutrition through fixation of gaseous N₂ from the atmosphere. For both NH₄⁺ and NO₃⁻-fed plants the N concentration in the plant tissues, particularly water-extractable NO₃⁻, increased at high supply concentrations, and concentrations of mineral cations generally decreased. It is concluded that S. sesban can grow without an external inorganic N supply by fixing atmospheric N₂ gas via root nodules. Also, S. sesban grows well on both NH₄⁺ and NO₃⁻ as the external N source and the plant can tolerate relatively high concentrations of NH₄⁺. This wide ecological amplitude concerning N nutrition makes S. sesban very useful as a N₂-fixing fallow crop in N deficient areas and also a candidate species for use in constructed wetland systems for the treatment of NH₄⁺ rich waters.     

NUTRIENT CONTROLS ON NITROGEN UPTAKE AND METABOLISM BY NATURAL POPULATIONS AND CULTURES OF TRICHODESMIUM (CYANOBACTERIA)

The effects of inorganic nutrient (ammonium [NH₄ ⁺ ] and nitrate [NO₃ ⁻ ]) and amino acid (glutamate [glu] and glutamine [gln]) additions on rates of N₂ fixation, N uptake, glutamine synthetase (GS) activity, and concentrations of intracellular pools of gln and glu were examined in natural and cultured populations of Trichodesmium. Additions of 1 μM glu, gln, NO₃ ⁻ , or NH₄ ⁺ did not affect short-term rates of N₂ fixation. This may be an important factor that allows for continued N₂ fixation in oligotrophic areas where recycling processes are active. N₂ fixation rates decreased when nutrients were supplied at higher concentrations (e.g. 10 μM). Uptake of combined N (NH₄ ⁺ , NO₃ ⁻ , and amino acids) by Trichodesmium was stimulated by increased concentrations. For NO₃ ⁻ , proportional increases in NO₃ ⁻ uptake and decreases in N₂ fixation were observed when additions were made to cultures before the onset of the light period. GS activity did not change much in response to the addition of NH₄ ⁺ , NO₃ ⁻ , glu, or gln. GS is necessary for N metabolism, and the bulk of this enzyme pool may be conserved. Intracellular pools of glu and gln varied in response to 10 μM additions of NH₄ ⁺ , glu, or gln. Cells incubated with NH₄ ⁺ became depleted in intracellular glu and enriched with intracellular gln. The increase in the gln/glu ratio corresponded to a decrease in the rate of N₂ fixation. Although the gln/glu ratio decreased in cells exposed to the amino acids, there was only a corresponding decrease in N₂ fixation after the gln addition. The results presented here suggest that combined N concentrations on the order of 1 μM do not affect rates of N₂ fixation and metabolism, although higher concentrations (e.g. 10 μM) can. Moreover, these effects are exerted through products of NH₄ ⁺ assimilation rather than exogenous N, as has been suggested for other species. These results may help explain how cultures of Trichodesmium are able to simultaneously fix N₂ and take up NH₄ ⁺ and how natural populations continue to fix N₂ once combined N concentrations increase within a bloom.     

Nitrogen metabolism by heterotrophic bacterial assemblages in Antarctic coastal waters

Field studies to examine the in situ assimilation and production of ammonium (NH₄ ⁺) by bacterial assemblages were conducted in the northern Gerlache Strait region of the Antarctic Peninsula. Short term incubations of surface waters containing ¹⁵N-NH₄ ⁺ as a tracer showed the bacterial population taking up 0.041–0.128 g-atoms Nl^–1d^–1, which was 8–25% of total NH₄ ⁺ uptake rates. The large bacterial uptake of NH₄ ⁺ occurred even at low bacterial abundance during a rich phytoplankton bloom. Estimates of bacterial production using ³H-leucine and -adenine were l.0gCl^–1 d^–1 before the bloom and 16.2 g Cl^–1 d^–1 at the bloom peak. After converting bacterial carbon production to an estimate of nitrogen demand, NH₄ ⁺ was found to supply 35–60% of bacterial nitrogen requirements. Bacterial nitrogen demand was also supported by dissolved organic nitrogen, generally in the form of amino acids. It was estimated, however, that 20–50% of the total amino acids taken up were mineralized to NH₄ ⁺. Bacterial production of NH₄ ⁺ was occurring simultaneously to its uptake and contributed 27–55% of total regenerated NH₄ ⁺ in surface waters. Using a variety of ¹⁵N-labelled amino acids it was found that the bacteria metabolized each amino acid differently. With their large mineralization of amino acids and their relatively low sinking rates, bacteria appear to be responsible for a large portion of organic matter recycling in the upper surface waters of the coastal Antarctic ecosystem.     

MACROALGAL RESPONSES TO NITROGEN SOURCE AND AVAILABILITY: AMINO ACID METABOLIC PROFILING AS A BIOINDICATOR USING GRACILARIA EDULIS (RHODOPHYTA)1

The use of macroalgae as biological indicators of dissolved nutrient source and availability in the water column was investigated. Total tissue nitrogen (N) content, pigments, and amino acids of the red alga Gracilaria edulis (Gmelin) Silva were compared to N source and availability in laboratory and field incubations to identify responses that would serve as bioindicators of N. Field-collected algae were preincubated (6–8 wk) in low-nutrient seawater to deplete their luxury reserves ofN. Incubations were then conducted for periods of 3 d in laboratory aquaria (N-spiked seawater) and in the field using macroalgal incubation chambers. After incubation in different N sources (NH₄⁺, NO₃^?, and urea) in laboratory aquaria, photosynthetic pigments (phycoerythrin and chlorophyll a) and total tissue N increased, in response to increasing [NH₄⁺] but not to [NO₃^?] or [urea]. Incubation in two ranges of [NH₄⁺], one from 0 to 80 μM and the other from 0 to 800 μM, in laboratory aquaria increased the total amino acid pool. Citrulline concentrations were the most responsive to [NH₄⁺] (r²= 0. 84). NH₄⁺ source treatments produced increases in citrulline, phenylalanine, serine, and free NH₄⁺ and decreases in alanine; NO₃^? treatments produced increases in glutamic acid, citrulline, and alanine; and urea treatments produced increases in free NH₄⁺ and decreases in phenylalanine and serine. The observed variations in amino acid content facilitated the development of an index for each N source based on relative concentrations of various amino acids (i. e. metabolic profiling). Gracilaria edulis was incubated along a field N gradient in the Brisbane River (three sites) and Moreton Bay (four sites), Queensland, Australia. Both phycoerythrin and tissue N appeared to respond equally to NH₄⁺ and NO₃^? availability in the field. N source indices, based on amino acid concentration, were effective predictors of both [NH₄⁺] and [NO₃^?] over a wide range of concentrations along the field gradient. Macroalgal physiological responses, particularly amino acid content, to changes in source and availability of N appear to be useful as sensitive bioindicators of N.     

Solutes contributing to osmotic adjustment in cultured plant cells adapted to water stress

Osmotic adjustment was studied in cultured cells of tomato (Lycopersicon esculentum Mill cv VFNT-Cherry) adapted to different levels of external water potential ranging from −4 bar to −28 bar. The intracellular concentrations of reducing sugars, total free amino acids, proline, malate, citrate, quaternary ammonium compounds, K⁺, NO₃⁻, Na⁺, and Cl⁻ increased with decreasing external water potential. At any given level of adaptation, the maximum contribution to osmotic potential was from reducing sugars followed by potassium ions. The sucrose levels in the cells were 3- to 8-fold lower than reducing sugar levels and did not increase beyond those observed in cells adapted to −16 bar water potential. Concentrations of total free amino acids were 4- to 5-fold higher in adapted cells. Soluble protein levels declined in the adapted cell lines, but the total reduced nitrogen was not significantly different after adaptation. Uptake of nitrogen (as NH₄⁺ or NO₃⁻) from the media was similar for adapted and unadapted cells. Although the level of quaternary ammonium compounds was higher in the nonadapted cells than that of free proline, free proline increased as much as 500-fold compared to only a 2- to 3-fold increase observed for quaternary ammonium compounds. Although osmotic adjustment after adaptation was substantial (up to −36 bar), fresh weight (volume increase) was restricted by as much as 50% in the adapted cells. Altered metabolite partitioning was evidenced by an increase in the soluble sugars and soluble nitrogen in adapted cells which occurred at the expense of incorporation of sugar into cell walls and nitrogen into protein. Data indicate that the relative importance of a given solute to osmotic adjustment may change depending on the level of adaptation.     

Simultaneous measurement of ammonium,nitrate and proton fluxes along the length of eucalypt roots

Knowledge of the preferred source of N for Eucalyptus nitens will lead to improved fertiliser management practices in plantations. Ion selective microelectrodes were used non-invasively to measure simultaneously net fluxes of NH₄ ⁺, NO₃ ^– and H⁺ along the tap root of solution-cultured E. nitens. Measurements were conducted in solutions containing 100 m NH₄NO₃. The pattern of fluxes was such that there was a large influx of NH₄ ⁺, a smaller influx of NO₃ ^– and large H⁺ efflux. The ratio of these fluxes was constant, according to the ratio 3:1:–6 (NH₄ ⁺:NO₃ ^–:H⁺). Within the region 20–60 mm from the root apex of E. nitens seedlings there was spatial and temporal variation in fluxes but flux patterns remained constant. Root hair density did not affect fluxes nor did proximity to lateral roots. Variation was less than that found in previous studies of localised root fluxes using similar high-resolution measurement techniques. It was concluded that small-scale spatial variation in fluxes may have confounded previous studies. There were associations between fluxes of all three ions, the strongest associations being between NH₄ ⁺ and H⁺, and NH₄ ⁺ and NO₃ ^–. Overall, these results are consistent with NH₄ ⁺ being the preferred source N for E. nitens.     

Organic and inorganic nitrogen nutrition of western red cedar,western hemlock and salal in mineral N-limited cedar–hemlock forests

Western red cedar (Thuja plicata Donn.), western hemlock (Tsuga heterophylla Raf. Sarge) and salal (Gaultheria shallon Pursh) are the main species growing in cedar–hemlock forests on Vancouver Island, Canada. Based on the dominance of organic N in these systems, we tested the hypotheses that: (1) organic N can be utilized by the three plant species; and (2) salal, which is ericoid mycorrhizal and has high tannin concentration in its tissues, would absorb more N from the complex organic N compounds than the other two species. The abilities of cedar, hemlock and salal to take up ¹⁵N,¹³C-labelled glutamic acid were measured and the capacities of the three species to use nitrate (NO₃^–), ammonium (NH₄⁺), glutamic acid, protein and protein–tannin N were compared over a 20-day period. Based on ¹³C enrichment, all three species absorbed at least a portion of glutamic acid intact. Cedar, hemlock and salal also showed similar patterns of N uptake from the NO₃^–, NH₄⁺, glutamic acid, protein and protein–tannin treatments. The largest proportions of applied N were taken up from the NO₃^– and NH₄⁺ treatments while smaller amounts of N were absorbed from the organic N compounds. Thus organic N was accessed to a modest degree by all three species, and salal did not have a greater capacity to utilize protein and protein–tannin–N.     

CHANGES IN INTRACELLULAR NITROGEN POOLS AND FEEDBACK CONTROLS ON NITROGEN UPTAKE IN CHAETOMORPHA LINUM (CHLOROPHYTA)1

Changes in the size of intracellular nitrogen pools and the potential feedback by these pools on maximum N uptake (NH₄⁺ and NO₃^?) rates were determined for Chaetomorpha linum (Müller) Kützing grown sequentially under nutrient-saturating and nutrient-limiting conditions. The size of individual pools in N-sufficient algae could be ranked as residual organic N (RON) comprised mainly of amino acids and amino compounds > protein N > NO₃^? > NH₄⁺ > chlorophyll N. When the external N supply was removed, growth rates remained high and individual N pools were depleted at exponential rates that reflected both dilution of existing pools by the addition of new biomass from growth and movement between the pools. Calculated fluxes between the tissue N pools showed that the protein pool increased throughout the N depletion period and thus did not serve a storage function. RON was the largest storage reserve; nitrate was the second largest, but more temporary, storage pool that was depleted within 10 days. Upon N resupply, the RON pool increased 3 × faster than either the inorganic or protein pools, suggesting that protein synthesis was the rate-limiting step in N assimilation and caused a buildup of intermediate storage compounds. Maximum uptake rates for both NH₄⁺ and NO₃^? varied inversely with macroalgal N status and appeared to be controlled by changes in small intracellular N pools. Uptake of NO₃^? showed an initial lag phase, but the initial uptake of NH₄⁺ was enhanced and was present only when the intracellular NH₄⁺ pool was depleted in the absence of an external N supply. A strong negative correlation between the RON pool size and maximum assimilation uptake rates for both NH₄⁺ and NO₃^? suggested a feedback control on assimilation uptake by the buildup and depletion of organic compounds. Enhanced uptake and the accumulation of N as simple organic compounds or nitrate both provide a temporary mechanism to buffer against the asynchrony of N supply and demand in C. linum.     

The influence of nitrogen concentration and ammonium/nitrate ratio on N-uptake,mineral composition and yield of citrus

In short-term water culture experiments with different ¹⁵N labeled ammonium or nitrate concentrations, citrus seedlings absorbed NH₄ ⁺ at a higher rate than NO₃ ^–. Maximum NO₃ ^– uptake by the whole plant occurred at 120 mg L^–1 NO₃ ^–-N, whereas NH₄ ⁺ absorption was saturated at 240 mg L^–1 NH₄ ⁺-N. ¹⁵NH₄ ⁺ accumulated in roots and to a lesser degree in both leaves and stems. However, ¹⁵NO₃ ^– was mostly partitioned between leaves and roots.Adding increasing amounts of unlabeled NH₄ ⁺ (15–60 mg L^–1 N) to nutrient solutions containing 120 mg L^–1 N as ¹⁵N labeled nitrate reduced ¹⁵NO₃ ^– uptake. Maximum inhibition of ¹⁵NO₃ ^– uptake was about 55% at 2.14 mM NH₄ ⁺ (30 mg L^–1 NH₄ ⁺-N) and it did not increase any further at higher NH₄ ⁺ proportions.In a long-term experiment, the effects of concentration and source of added N (NO₃ ^– or NH₄ ⁺) on nutrient concentrations in leaves from plants grown in sand were evaluated. Leaf concentration of N, P, Mg, Fe and Cu were increased by NH₄ ⁺ versus NO₃ ^– nutrition, whereas the reverse was true for Ca, K, Zn and Mn.The effects of different NO₃ ^–-N:NH₄ ⁺-N ratios (100:0, 75:25, 50:50, 25:75 and 0:100) at 120 mg L^–1 total N on leaf nutrient concentrations, fruit yield and fruit characteristics were investigated in another long-term experiment with plants grown in sand cultures. Nitrogen concentrations in leaves were highest when plants were provided with either NO₃ ^– or NH₄ ⁺ as a sole source of N. Lowest N concentration in leaves was found with a 75:25 NO₃ ^–-N/NH₄ ⁺-N ratio. With increasing proportions of NH₄ ⁺ in the N supply, leaf nutrients such as P, Mg, Fe and Cu increased, whereas Ca, K, Mn and Zn decreased. Yield in number of fruits per tree was increased significantly by supplying all N as NH₄ ⁺, although fruit weight was reduced. The number of fruits per tree was lowest with the 75:25 NO₃ ^–-N:NH₄ ⁺-N ratio, but in this treatment fruits reached their highest weight. Rind thickness, juice acidity, and colour index of fruits decreased with increasing NH₄ ⁺ in the N supply, whereas the % pulp and maturity index increased. Percent of juice in fruits and total soluble solids were only slightly affected by NO₃ ^–:NH₄ ⁺ ratio.     

Growth and solute composition of the salt-tolerant kallar grass [Leptochloa fusca (L.) Kunth] as affected by nitrogen source

A study was conducted to elucidate the effect of N form, either NH₄ ⁺ or NO₃ ^–, on growth and solute composition of the salt-tolerant kallar grass [Leptochloa fusca (L.) Kunth] grown under 10 mM or 100 mM NaCl in hydroponics. Shoot biomass was not affected by N form, whereas NH₄ ⁺ compared to NO₃ ^– nutrition caused an almost 4-fold reduction in the root biomass at both salinity levels. Under NH₄ ⁺ nutrition, salinity had no effect on the biomass yield, whereas under NO₃ ^– nutrition, increasing salinity from 10 mM to 100 mM caused 23% and 36% reduction in the root and shoot biomass, respectively. The reduced root growth under NH₄ ⁺ nutrition was not attributable to impaired shoot to root C allocation since N form did not affect the overall root sugar concentration and the starch concentration was even higher under NH₄ ⁺ compared to NO₃ ^– nutrition. The low NH₄ ⁺ (2 mM) and generally higher amino-N concentrations in NH₄ ⁺- compared to NO₃ ^–-fed plants indicated that the grass was able to effectively detoxify NH₄ ⁺. Salinity had no effect on Ca²⁺ and Mg²⁺ levels, whereas their concentration in shoots was lower under NH₄ ⁺ compared to NO₃ ^– nutrition (over 66% reduction in Ca²⁺; over 20% reduction in Mg²⁺), but without showing deficiency symptoms. Ammonium compared to NO₃ ^– nutrition did not inhibit K⁺ uptake, and the K⁺-Na⁺ selectivity either remained unaffected or it was higher under NH₄ ⁺ than under NO₃ ^– nutrition. Results suggested that while NH₄ ⁺ versus NO₃ ^– nutrition substantially reduced root growth, and also strongly modified anion concentrations and to a minor extent concentrations of divalent cations in shoots, it did not influence salt tolerance of kallar grass.     

The differing responses of two <Emphasis Type="Italic">Arabidopsis</Emphasis> ecotypes to ammonium are modulated by the photoperiod regime

Responses to excessive ammonium (NH₄ ⁺) were compared between two Arabidopsis ecotypes (Col-0, JA22) with respect to different photoperiods in hydroponics. In this study, we showed that external extra NH₄ ⁺ led to severe growth suppression, accumulations of free NH₄ ⁺ and amino acids and increased the activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in shoots of the two Arabidopsis ecotypes. However, the levels of free NH₄ ⁺ and total amino acids increased, whereas the activities of GS, NADH-dependent glutamate synthase and GDH decreased under the continuous light when compared with the light (16 h)–dark (8 h) cycle photoperiod. Statistical analyses suggested that strong correlations exist among the growth reduction, accumulations of free NH₄ ⁺, total amino acids and levels of GS activity in shoots under the high NH₄ ⁺ stress regardless of the photoperiod regimes. Interestingly, under the continuous light, Col-0 showed more resistant to such growth reduction and maintained about onefold higher capability of converting excess free NH₄ ⁺ into amino acids, with onefold higher GS activity induced by the external NH₄ ⁺ when compared with JA22. In contrast, these differences were abolished between Col-0 and JA22 under the light–dark cycle condition. Taken together, our results conclude that the sensitivity to NH₄ ⁺ of Col-0 and JA22 is changed between the continuous light and the light–dark cycle photoperiod, which is correlative to the alteration of the GS activity in shoots.     

Nitrate Reduction in a Groundwater Microcosm Determined by 15N Gas Chromatography-Mass Spectrometry

Aerobic and anaerobic groundwater continuous-flow microcosms were designed to study nitrate reduction by the indigenous bacteria in intact saturated soil cores from a sandy aquifer with a concentration of 3.8 mg of NO₃⁻-N liter⁻¹. Traces of ¹⁵NO₃⁻ were added to filter-sterilized groundwater by using a Darcy flux of 4 cm day⁻¹. Both assimilatory and dissimilatory reduction rates were estimated from analyses of ¹⁵N₂, ¹⁵N₂O, ¹⁵NH₄⁺, and ¹⁵N-labeled protein amino acids by capillary gas chromatography-mass spectrometry. N₂ and N₂O were separated on a megabore fused-silica column and quantified by electron impact-selected ion monitoring. NO₃⁻ and NH₄⁺ were analyzed as pentafluorobenzoyl amides by multiple-ion monitoring and protein amino acids as their N-heptafluorobutyryl isobutyl ester derivatives by negative ion-chemical ionization. The numbers of bacteria and their [methyl-³H]thymidine incorporation rates were simultaneously measured. Nitrate was completely reduced in the microcosms at a rate of about 250 ng g⁻¹ day⁻¹. Of this nitrate, 80 to 90% was converted by aerobic denitrification to N₂, whereas only 35% was denitrified in the anaerobic microcosm, where more than 50% of NO₃⁻ was reduced to NH₄⁺. Assimilatory reduction was recorded only in the aerobic microcosm, where N appeared in alanine in the cells. The nitrate reduction rates estimated for the aquifer material were low in comparison with rates in eutrophic lakes and coastal sediments but sufficiently high to remove nitrate from an uncontaminated aquifer of the kind examined in less than 1 month.     

Dissimilatory Reduction of NO2− to NH4+ and N2O by a Soil Citrobacter sp.

Dissimilatory reduction of NO₂⁻ to N₂O and NH₄⁺ by a soil Citrobacter sp. was studied in an attempt to elucidate the physiological and ecological significance of N₂O production by this mechanism. In batch cultures with defined media, NO₂⁻ reduction to NH₄⁺ was favored by high glucose and low NO₃⁻ concentrations. Nitrous oxide production was greatest at high glucose and intermediate NO₃⁻ concentrations. With succinate as the energy source, little or no NO₂⁻ was reduced to NH₄⁺ but N₂O was produced. Resting cell suspensions reduced NO₂⁻ simultaneously to N₂O and free extracellular NH₄⁺. Chloramphenicol prevented the induction of N₂O-producing activity. The K_m for NO₂⁻ reduction to N₂O was estimated to be 0.9 mM NO₂⁻, yet the apparent K_m for overall NO₂⁻ reduction was considerably lower, no greater than 0.04 mM NO₂⁻. Activities for N₂O and NH₄⁺ production increased markedly after depletion of NO₃⁻ from the media. Amendment with NO₃⁻ inhibited N₂O and NH₄⁺ production by molybdate-grown cells but not by tungstate-grown cells. Sulfite inhibited production of NH₄⁺ but not of N₂O. In a related experiment, three Escherichia coli mutants lacking NADH-dependent nitrite reductase produced N₂O at rates equal to the wild type. These observations suggest that N₂O is produced enzymatically but not by the same enzyme system responsible for dissimilatory reduction of NO₂⁻ to NH₄⁺.     

Growth and Nitrogen Uptake Kinetics in Cultured Prorocentrum donghaiense

We compared growth kinetics of Prorocentrum donghaiense cultures on different nitrogen (N) compounds including nitrate (NO₃ ⁻), ammonium (NH₄ ⁺), urea, glutamic acid (glu), dialanine (diala) and cyanate. P. donghaiense exhibited standard Monod-type growth kinetics over a range of N concentraions (0.5–500 μmol N L⁻¹ for NO₃ ⁻ and NH₄ ⁺, 0.5–50 μmol N L⁻¹ for urea, 0.5–100 μmol N L⁻¹ for glu and cyanate, and 0.5–200 μmol N L⁻¹ for diala) for all of the N compounds tested. Cultures grown on glu and urea had the highest maximum growth rates (μ_m, 1.51±0.06 d⁻¹ and 1.50±0.05 d⁻¹, respectively). However, cultures grown on cyanate, NO₃ ⁻, and NH₄ ⁺ had lower half saturation constants (K_μ, 0.28–0.51 μmol N L⁻¹). N uptake kinetics were measured in NO₃ ⁻-deplete and -replete batch cultures of P. donghaiense. In NO₃ ⁻-deplete batch cultures, P. donghaiense exhibited Michaelis-Menten type uptake kinetics for NO₃ ⁻, NH₄ ⁺, urea and algal amino acids; uptake was saturated at or below 50 μmol N L⁻¹. In NO₃ ⁻-replete batch cultures, NH₄ ⁺, urea, and algal amino acid uptake kinetics were similar to those measured in NO₃ ⁻-deplete batch cultures. Together, our results demonstrate that P. donghaiense can grow well on a variety of N sources, and exhibits similar uptake kinetics under both nutrient replete and deplete conditions. This may be an important factor facilitating their growth during bloom initiation and development in N-enriched estuaries where many algae compete for bioavailable N and the nutrient environment changes as a result of algal growth.     

Nitrogen relations of a low nitrate uptake inbred line of white clover (Trifolium repens L.)

The nitrogen relations of an inbred line of white clover (Trifolium repens L.) thought to exhibit an abnormally low capacity for NO₃ ^– uptake (line LNU) were compared with a line regarded as normal with respect to NO₃ ^– uptake (line NNU). Growth, nodulation, N₂ fixation and NO₃ ^– uptake were measured over 7 weeks in flowing solution culture (Experiment 1) by plants dependent for N acquisition on either (i) NO₃ ^– uptake, (ii) NO₃ ^– uptake +N₂ fixation, or (iii) N₂ fixation only. Effects of plant N status on the short-term uptake and translocation of ¹⁵NH₄ ⁺ and ¹⁵NO₃ ^– were also investigated (Experiment 2). Nitrate uptake per plant by –fix/+NO₃ ^– line LNU was 50% of uptake by line NNU over 35 days, and there were significant differences in specific uptake rates of NO₃ ^– between the lines over the first 24 days. The `low NO<sub>3</sub> <sup>–</sup> uptake' phenotype was indistinct under +fix/+NO<sub>3</sub> <sup>–</sup> treatment. Nitrate lowered specific rates of nitrogen fixation by line NNU but had no effect on line LNU. Only low N status line LNU plants had lower short-term rates of NH<sub>4</sub> <sup>+</sup> and NO<sub>3</sub> <sup>–</sup> uptake than line NNU. It is concluded that the `low NO₃ ^– uptake' phenotype of line LNU is inconsistently expressed. Circumstantial evidence points to increased NO₃ ^– efflux and decreased xylem translocation of NO₃ ^– as possible explanations for the lower NO₃ ^– uptake by line LNU.     

In vitro weathering of phlogopite by ectomycorrhizal fungi

Oxalate accumulation in external medium under hyphal mats of two ectomycorrhizal species is strongly stimulated (1.7 to 35 fold) by a simultaneous depletion of available K⁺ and Mg²⁺. Pisolithus tinctorius strain 441 accumulates oxalate both on NH₄–N and on NO₃–N whereas Paxillus involutus strain COU only accumulates oxalate on NO₃–N. On NO₃–N, under a simultaneous K⁺ and Mg²⁺ deficiency, P. involutus COU is a very active oxalate producer compared to P. tinctorius 441. The present results could explain the various mineralogical evolutions of a phlogopite mica previously recorded under P. involutus COU or P. tinctorius 441 and suggest a key role for fungal oxalic acid during mineral weathering in response to nutrient deficiency.     

Changing nitrogen source on the yield and nitrogen content of soybeans

Summary Soybean plants were grown in nutrient culture solutions containing 150 ppm of N either as an equal concentration of NH₄ ⁺ or NO₃ ^–, or all NO₃ ^–. At the R2 stage of growth for some plants, the N form was changed to either all NO₃ ^– or all NH₄ ⁺, but at the same total N concentration as before. Highest seed yield was obtained with all NO₃ ^– over the entire growth period, the poorest when the N form was switched from an equal ratio of NH₄ ⁺ and NO₃ ^– to all NH₄ ⁺ at the R2 stage. Kjeldahl N concentrations in the plant leaves and seed were highest when NH₄ ⁺ was part or all of the N source in the nutrient solution. These results may partially explain why the literature is inconsistent on the effect of added fertilizer N on soybean seed yield, and may pose a problem in using leaf Kjeldahl N concentration to determine plant N sufficiency.     

Nitrous Oxide Emission Associated with Autotrophic Ammonium Oxidation in Acid Coniferous Forest Soil

Aerobic N₂O production was studied in nitrifying humus from urea-fertilized pine forest soil. Acetylene and nitrapyrin inhibited both NH₄⁺ oxidation and N₂O production, indicating that N₂O production was closely associated with autotrophic NH₄⁺ oxidation. N₂O production was enhanced by low soil pH; it was negligible above pH 4.7. When soil pH decreased from 4.7 to 4.1, the relative amount of N₂O-N produced from NH₄⁺-N oxidized increased exponentially to 20%. There was also some evidence that N₂O formation was stimulated by salts (potassium sulfate and sodium phosphates). The maximum rate of N₂O-N production was 0.17 μg of N₂O-N per g of soil per h. When humus was treated with NO₂⁻, N₂O evolved immediately, indicating chemical formation, but no N₂O was formed on the addition of NO₃⁻. The amount of N₂O-N evolved was 0.6 to 4.6% of NO₂⁻-N added. A high concentration of NO₂⁻ and low soil pH enhanced chemical production of N₂O. There was no accumulation of NO₂⁻ during nitrification. The calculations indicated that chemical formation of N₂O was not the main source of N₂O during NH₄⁺ oxidation. After the addition of inhibitors of NH₄⁺ oxidation the soils contained NO₃⁻, but no N₂O was produced. The results suggest that enhanced autotrophic NH₄⁺ oxidation is a potential source of N₂O in fertilized acid forest soil.