Nitrate and nitrite reduction by alfalfa root nodules: Accumulation of nitrite in Rhizobium melioti bacteroids and senescence of nodules

Addition of NO⁻₃ rapidly induced senescence of root nodules in alfalfa ( Medicago sativa L. cv. Aragon). Loss of nodule dry matter began at the lowest NO⁻₃ concentration (10 m M ) but degradation of bacteroid proteins was only detected when nodules were supplied with NO⁻₃ concentrations above 20 m M .
Bacteroids from Rhizobium meliloti contained high specific activities of nitrate reductase (NR) and nitrite reductase (NiR). Both enzymes were presumably substrate-induced although substantial enzyme activities were present in the absence of NO⁻₃ Typical specific activities for soluble NR and NiR of bacteroids under NO⁻₃ free conditions were 1.2 and 1.4 μmol (mg protein)⁻¹h⁻¹, respectively. In the presence of NO⁻₃, the specific activity of NR was considerably greater than that of NiR, thus causing NO⁻₂ accumulation in bacteroids. Nitrite levels in the bacteroids were linearly correlated with specific activities of NR and NiR, indicating that NO⁻₂ is formed by bacteroid NR and that this NO⁻₂ in turn, induces bacteroid NiR. Accumulation of NO⁻₂ within bacteroids also indicates that NO⁻₂ inhibits nodule activity after feeding plants with NO⁻₃     

Nitrate reductase and molybdenum cofactor in annual ryegrass as affected by salinity and nitrogen source

The influence of salinity on the activity of nitrate reductase (NR, EC 1.6.6.1) and the level of the molybdenum cofactor (MoCo) as affected by the source and concentration of nitrogen was studied in annual ryegrass ( Lolium multiflorum cv. Westerwoldicum). Plants grown in sand were irrigated with nutrient solution with an electrical conductivity of 2 or 11.2 dS m⁻¹, containing nitrogen (0.5 or 4.5 m M ) in the form of NH₄NO₃ or NaNO₃ Salinity-treated (11.2 dS m⁻¹) plants produced less biomass and more organic nitrogen while accumulating more NO⁻₃ than control plants. Increased nitrogen concentration in the irrigation solutions enhanced biomass and organic nitrogen production as well as NO⁻₃ accumulation irrespective of the electrical conductivity. Salinity inhibited shoot growth and increased shoot NR activity of plants receiving 4.5 m M NH₄NO₃ or NaNO₃. Similar effects were observed in roots of plants grown in 4.5 m M NaNO₃. Nitrate added to a complementation medium containing ryegrass MoCo and the NR apoprotein of Neurospora crassa mutant nit-1 stimulated the activity of the reconstituted NR (NADPH-nitrate reductase, EC 1.6.6.3). Increased salinity and nitrogen in the nutrient solutions caused an increase of MoCo content in roots and shoots. Similar results were observed for NR activity in the shoots. The increase of MoCo in response to salinity was more pronounced than that of NR, especially in the roots. We conclude that the pool size of MoCo in ryegrass is not constant, but varies in response to nutritional and environmental factors.     

Nitrite in the root zone and its effects on ion uptake and growth of wheat seedlings

The immediate and posteffects of various concentrations of NaNO₂ on ion uptake of wheat ( Triticum aestivum L. cv. GK Öthalom) seedlings were studied at different pH values. Without pretreatment, the higher the concentration of NaNO₂ the greater was the decrease in uptake of K⁺ into the roots, both at pH 4 and pH 6. At pH 6 but not at pH 4 the reverse was true when the seedlings were pretreated with NaNO₂. Due to the high Na⁺ content of the roots, an effect of Na⁺ in this process cannot be excluded. Nitrite was taken up by the roots more rapidly than nitrate. Nitrite at 0.1 m M in the medium induced the development of an uptake system for both NO⁻₂ and NO⁻₃ in wheat roots. At higher concentrations pretreatment with NO⁻₂ decreased NO⁻₃ uptake by the roots, but NO₃ did not inhibit the uptake of NO₂. The toxic effect of NO⁻₂ was strongly pH dependent. Lower pH of the external solution led to an increased inhibition by NO⁻₂ of both ion uptake and growth of seedlings. The inhibitory effect of NO⁻₂ differed considerably for roots and shoots. The roots and especially the root hairs were particularly sensitive to NO⁻₂ treatment.     

Uptake regions of inorganic nitrogen in roots of carob seedlings

Three-week-old seedlings of carob ( Ceratonia siliqua L. cv. Mulata) were grown for 9 weeks under different root temperatures (20, 30 and 40°C) at pH values of 5, 7 and 9 with nitrate or ammonium as nitrogen source. Nitrogen uptake rates were determined by depletion from the medium and decreased with distance from the apex. The decline of nitrogen uptake rates along the roots depended on the form of inorganic nitrogen in the medium as well as on pH and temperature, such that the NO⁻₃ and NH⁺₄ ions were taken up essentially by the root tips (0–2 cm) through processes requiring energy. The uncharged NH₃ species entered passively, through the mature parts of the root (2–10 cm). Root zone temperature and pH affect the NH⁺₄/NH³ equilibrium in the nutrient solution and, consequently, the uptake areas of the root for these ions. Furthermore. while root tip uptake of nitrogen is energy dependent, uptake through mature root areas is essentially passive and seems to depend on a well developed apparent free space.     

Effects of sucrose on betacyanin accumulation and growth in suspension cultures of Phytolacca americana

The effects of sucrose on betacyanin accumulation and growth in suspension cultures of Phytolacca americana L. were investigated. Maximal betacyanin accumulation was observed at 88 m M sucrose on cell number basis and at 175 m M sucrose on fresh weight basis. This is because cell size decreased as the initial sucrose concentration was increased. Supplementary studies using mannitol indicated that sucrose itself caused increased cell number and that cell size was affected by both sucrose concentration and water potential. Betacyanin accumulation per cell and per fresh weight at a constant concentration of sucrose (88 m M ) decreased with decreasing water potential. When sucrose concentration increased at a constant water potential (–0.7 MPa), betacyanin accumulation per fresh weight increased up to 88 m M and remained at constant level at higher concentrations, while betacyanin accumulation per cell decreased remarkably, due to a dramatic increase in cell number.     

Effect of a constant supply of different nitrogen sources on protein and carbohydrate content and enzyme activities of Anabaena variabilis cells

The effect of the nitrogen source on carbohydrate and protein contents and on several enzymatic activities involved in the carbon and nitrogen metabolism was studied in Anabaena variabilis ATCC 29413 cells grown under a constant supply of either N, NO⁻₃ or NH⁺₄ at different concentrations. An enhancement of protein content accompanied by a parallel decrease of carbohydrates was observed with increasing NO⁻₃ or NH⁺₄ concentrations in the medium. In cultures containing 0.1 m M NO⁻₃ or 0.1 m M NH⁺₄ nitrogenase (EC 1.18.6.1) activity was 74 and 66%, respectively, of that found in N₂-grown cells. This activity was still present with 1 m M NO⁻₃ or 1 m M NH⁺₄ in the medium and even with 10 m M NO⁻₃, but it was completely inhibited by 5 m M NH⁺₄. Ferredoxin-nitrate reductase (EC 1.7.7.2) activity was detected only in NO⁻₃ grown cells and simultaneously with nitrogenase activity. Increasing concentrations of combined nitrogen in the medium, especially NH⁺₄, promoted a concomitant decline of glutamine synthetase (EC 6.3.1.2), NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42), and NAD⁺-malate dehydrogenase (EC 1.1.1.37) activities, suggesting that these enzymes play an important role in the regulation of carbon-nitrogen metabolism in cyanobacteria.     

Effects of NH+4-N and NO+3-N on growth and metabolism of Sphagnum magellanicum

Photosynthetic CO₂-fixation, chlorophyll content, growth rate and nitrate reductase activity were used to examine the influence of NH⁺₄-N and NO⁻₃-N on Sphagnum magellanicum cultivated under defined conditions in phytotrons. NO⁻₃-concentrations up to 322 μ M were found to be favourable. Increased NH⁺₄ concentrations, however, resulted in growth inhibition and decreased chlorophyll content at concentrations ≧ 255 μ M ; e.g. 600 μ M NH⁺₄ caused a 20% reduction of nitrate reductase activity and net photosynthesis. For raised bog Sphagna an improved standard nutrient solution is proposed with the following ion concentrations (μ M ): 55 Na⁺; 17 K⁺; 95 NH⁺₄; 22 Ca²⁺; 22 Mg²⁺; 2 Fe³⁺; 20 Cl⁻; 100 NO⁻₃; 57 SO^2-₄; 7.4 H₂PO⁻₄; trace elements: A-Z solution (Hoagland) 50 μl 1000 ml⁻¹; pH 5.8.     

Ammonium rhythm in cultures of the cyanobacterium Microcystis firma

Over a period of several days, rhythmic changes in extracellular NH⁺₄ concentration take place in cultures of the cyanobacterium Microcystis firma (Bré et Lenorm.) Schmidle, strain Gromov/St. Petersb. 398, under conditions of restricted CO₂ supply and light/dark alternation. The changes are enhanced by nitrate supply. Among the various processes generating intracellular NH⁺₄ (NH⁴₄ uptake, NO⁻₃ reduction, protein and amino acid degradation, photorespiration), NO⁻₃ reduction appears as the one most important. This can be concluded from experiments with and without nitrate and/or ammonium in the medium. In the presence of saturating CO₂, continuous light, or continuous darkness, rhythmic NH⁺⁴₄ oscillations are not induced. Studies of the incorporation of NH⁺₄ nitrogen by in vivo ¹⁵N-NMR show that if CO₂ is supplied, ¹⁵N is accumulated in several components with the following time course: in the first hour in Gln (δ), in the second hour in the α-amino groups of most nonbranched amino acids, in the third hour in γ-aminobutyric acid (GABA), Orn (δ) and Lys (ε), and in the sixth hour in Ala. Carbon limitation, however, results in accumulation of label in the amide nitrogen of glutamine only.     

Assimilation of nitrate and ammonium by the Scots pine (Pinus sylvestris) seedling under conditions of high nitrogen supply

Seedlings of Scots pine ( Pinus sylvestris L.) were grown on perlite for 21 days under controlled conditions. Apart from the water control, KNO₃ (15 m M ), (NH₄)₂SO₄ (7.5 m M ), and NH₄NO₃ (15 m M ) were offered to study the effects of a high nitrogen supply on nitrogen assimilation. In some experiments 1.3 m M potassium was added to the basic ammonium solutions. In labelling studies nitrate and ammonium were 2.3 atom%¹⁵N-enriched. It was found that over the 21-day period approximately three times more ammonium-N was taken up than nitrate-N. However, nitrate and ammonium, applied simultaneously, were taken up to the same extent as if they were applied separately (additivity). The presence of K⁺ in the medium did not affect N-uptake. Among the soluble N-containing compounds nitrate, ammonium and 8 amino acids were quantified. It was found that assimilation of nitrate can cope with the uptake of NO⁻₃ under all circumstances. Neither free nitrate nor ammonium or amino acids accumulated to an extent exceeding the values of water-grown seedlings. On the other hand, in case of high ammonium supply considerably more nitrogen was taken up than could be incorporated into nonsoluble N-containing substance ('protein'). The remaining nitrogen was found to accumulate in intermediary storage pools (free NH₄⁺, glutamine, asparagine, arginine). Part of this accumulated N could be incorporated into protein when potassium was offered in the nutrient solution. It is concluded that potassium is a requirement for a high rate of protein synthesis not only in crop plants but also in conifers.     

The use of compound continuous flow diffusion chemostats to study the interaction between nitrifying and nitrate-reducing bacteria

Abstract The interactions occuring between populations of a nitrate-respiring Vibrio sp. and autotrophic nitrifying bacteria belonging to the genera Nitrosomonas and Nitrobacter have been investigated in a compound bi-directional flow diffusion chemostat at a dilution rate of 0.025 h⁻¹ and a temperature of 25°C. When grown under NO⁻₃ limitation, the Vibrio sp. produced NH⁺₄ as the principal end-product of nitrate respiration, and there was a corresponding significant increase in cell numbers of the Nitrosomonas sp. population, which derived energy by the oxidation of NH⁺₄ to NO⁻₂. Nitrite in turn was used by the Nitrobacter sp. population as an energy source with the concomitant regeneration of NO⁻₃. Under NO⁻₃ excess growth conditions the Vibrio sp. produced NO⁻₂ rather than NH⁺₄ as the major product of NO⁻₃ dissimilation, and growth of the Nitrobacter population was stimulated as increased quantities of NO⁻₂ became available. In contrast, the Nitrosomonas sp. population declined sharply as the energy source NH⁺₄ became limiting. These data demonstrate that defined mixed populations of obligately aerobic nitrifying bacteria and facultatively anaerobic nitrate respiring bacteria can co-exist for extended time periods and operate an internal nitrogen cycle which is energetically beneficial to both populations.     

Nitrate-induced de novo synthesis and regulation of NAD(P)H nitrate reductase from Sphagnum

The NO⁻₃-triggered induction of nitrate reductase (NR; EC 1.6.6.2) in the bryophyte Sphagnum magellanicum Brid. has been studied, using in vivo and in vitro assays as well as immunological methods. The time-course of induction was triphasic with maximal NR activity after 6–8 h. Results obtained from Western blots show that NR is synthesized de novo after NO⁻₃ application. The inhibitory effect of cycloheximide on NR induction corroborated this conclusion. Light enhanced the NO⁻₃-triggered NR induction. The enzyme activity, measured in vivo, increased more than the in vitro activity. No evidence for phytochrome control of NR was found. Nitrate uptake, in contrast to NR activity, showed no lag period after NO⁻₃ application and, under the experimental conditions used, was not rate limiting for NR induction. Neither light nor a NO⁻₃ pretreatment significantly affected NO⁻₃ uptake.     

The Effect of Potassium Nitrate on the Reduction of Phytophthora Stem Rot Disease of Soybeans, the Growth Rate and Zoospore Release of Phytophthora sojae

The effects of potassium nitrate (KNO₃) application on Phytophthora stem rot disease reduction of Glycine max (L.) Merr. cvs. Chusei-Hikarikuro and Sachiyutaka, and mycelium growth and zoospore release of a Phytophthora sojae isolate were investigated under laboratory conditions. The application of 4–30 m m KNO₃ prior to inoculation greatly reduced incidence of disease in the two soybean cultivars. Although a concentration of 20–30 m m KNO₃ led to a slight decrease in the growth rate of the PJ-H30 isolate on PDA medium, no significant relationship was observed between inhibition of the growth rate and disease reduction on application of 0.4–10 m m KNO₃. Disease suppression recorded in laboratory experiments using pathogen mycelium was due to the response of plant tissues rather than a direct inhibition of pathogen hyphal growth by the application of KNO₃. The extent of disease reduction was related to increased potassium concentration in plants of the two cultivars (except for some cases involving cv. Sachiyutaka), suggesting that differences existed between the two cultivars in terms of the effect of KNO₃ application on disease suppression. Scanning electron microscopic observation with fresh samples indicated marked accumulation of potassium at the penetration-stopping sites of P. sojae in the cortex layer of soybean plants treated with 30 m m KNO₃, compared with the non-treated control plants. The presence of 0.4–30 m m KNO₃ decreased the release of zoospores. These results suggest the possibility of applying a solution containing 20–30 m m of KNO₃ to decrease the incidence of disease in agricultural fields by the response of plant tissues to KNO₃.     

Effects of copper on active and passive Rb+ influx in roots of winter wheat

The effects of copper (CuCl₂) on active and passive Rb⁺(⁸⁶Rb⁺) influx in roots of winter wheat grown in water culture for 1 week were studied. External copper concentrations in the range of 10–500 μ M in the uptake nutrient solution reduced active Rb⁺ influx by 20–70%, while passive influx was unaffected (ca 10% of the Rb⁺ influx in the Cu-free solution). At external Rb⁺ concentrations of up to 1 m M , Cu exposure (50 μ M decreased V_max to less than half and increased K_m to twice the value of the control. Short Cu exposure reduced the K⁺ concentration in roots of low K⁺ status. Pretreatment for 5 min in 50 μ M CuCl₂ prior to uptake experiments reduced Rb⁺ influx by 26%. After 60 min pretreatment with Cu, the corresponding reduction was 63%. Cu in the cultivation solution impeded growth, especially of the roots. The Cu concentration in the roots increased linearly with external Cu concentration (0–100 μ M ) while Cu concentration in the shoots was relatively unchanged. The K⁺ concentration in both roots and shoots decreased significantly with increased Cu in the cultivation solutions. Possible effects of Cu on membranes and ion transport mechanisms are discussed.     

Relation of light and nitrogen source to growth, nitrate reductase and glutamine synthetase activity of jack pine seedlings

Two-month-old jack pine ( Pinus banksiana Lamb.) seedlings were placed in a greenhouse where both nitrogen source and light level were varied. After 4 months, whole seedling biomass, leaf biomass and relative growth rate were greatest in seedlings grown with NH⁺₄/NO/NO⁻₃-N and full light (FL) and least in seedlings grown with NO ⁻₃-N and low light (LL). NO ⁻₃-seedlings grown under full light and NH⁺₄/NO⁻₃-seedlings grown under low light were approximately equal. This indicates that the extra carbon costs of assimilating only NO⁻₃-N were similar to the reduction of carbon fixation resulting from a 50% decrease in photon flux density. Percentage and total nitrogen content of needles were greater in seedlings grown under low light independent of nitrogen fertilization. Percentage and total nitrogen content of roots were higher under low light and lower when fertilized with NO⁻₃.
Nitrate reductase (NR) activity was higher in roots than in needles, while glutamine synthetase (GS) activity was higher in needles than in roots. Low light resulted in decreased NR activity (mg N)⁻¹ in needles, but not in roots. However, no nitrate was detected in the needles in any treatment. GS activity, on the other hand, was greater under low light in both needles and roots. GS activity in needles is most likely involved with the reassimilation rather than the initial assimilation of ammonium. Some implications of these shifts in enzymatic activity for ecological phenomena in forests are discussed.     

Chromium-induced inhibition of ethylene evolution in bean (Phaseolus vulgaris) leaves

The influence of chromium concentration on ethylene production in bean plants ( Phaseolus vulgaris L. cv. Contender) was investigated. A Cr ion-induced inhibition of ethylene synthesis from endogenous 1-aminocyclopropane-1-carboxylic acid (ACC) was observed within both leaf discs floated on 2 m M CrO²⁻₄ or Cr³⁺ and leaf discs from plants cultured in nutrient solutions containing 10, 20 or 40 μ M CrO²⁻₄. However, Cr ions supplied either to plants with the nutrient solution or to discs with the incubation medium rather increased the conversion of exogenous ACC to ethylene. Primary leaves of plants exposed to CrO²⁻₄-containing nutrient solutions showed a statistically insignificant decrease of ACC-synthase activity. In the trifoliolate leaves of plants exposed to 10 μ M CrO²⁻₄, in which a significant decrease of ethylene production from endogenous ACC was observed, a substantial increase of ACC synthase was found. These results indicate that Cr ion-induced inhibition of ethylene production is not due to a breakdown of membrane integrity, which is necessary for ethylene forming enzyme activity, but caused by metabolic alterations leading to decreased ACC availability. Chromium ions may act by inhibiting ACC synthase activity or by diverting a metabolic step prior to the ACC synthase catalyzed reaction.     

Author idex volume 36 (1986)

Abstract Nitrate reduction to ammonia by marine Vibrio species was studied in batch and continuous culture. In pH-controlled batch cultures (pH 7.4; 50 mM glucose, 20 mM KNO₃), the nitrate consumed accumulated to more than 90% as nitrite. Under these conditions, the nitrite reductase (NO⁻₂→ NH₃) was severely repressed. In pH-controlled continuous cultures of V. alginolyticus with glucose or glycerol as substrates ( D = 0.045 h⁻¹) and limiting N-source (nitrate or nitrite), nitrite reductase was significantly derepressed with cellular activities in the range of 0.7–1.2 μmol min⁻¹ (mg protein)⁻¹. The enzyme was purified close to electrophoretic homogeneity with catalytic activity concentrations of about 1800 nkat/mg protein. It catalyzed the reduction of nitrite to ammonia with dithionite-reduced viologen dyes or flavins as electron donors, had an M _r of about 50 000 (determined by gel filtration) and contained c-type heme groups (probably 4–6 per molecule).     

Influence of nitrate and ammonium on the growth and 2,4-diaminobutyric acid composition of flatpea (Lathyrus sylvestris L.)

Abstract. Presence of 2.4-diaminobutyric acid (A₂bu), a neurotoxin, in tissues of flatpea ( Lathyrus sylvestris L.) necessitates a thorough understanding of the regulation of this nonprotein amino acid before the species can be recommended to livestock producers for forage applications. To determine how different concentrations and ratios of NO₃ and NH⁺₄ in growth media influence the levels of A₂bu and other free amino acids in the 'Lathco'flatpea cultivar, plants were grown hydroponically in controlled environments. The concentration of A₂bu was highest in tissues when the NO₃ to NH⁺₄ ratio in the nutrient solution was low. Responses of amides and other nonprotein amino acids, especially in the roots, followed a similar trend. Free protein amino acids in leaves and stems were generally unaffected by changes in NO₃ to NH⁺₄ ratios. In roots, protein amino acids increased as the NO₃ to NH⁺₄ ratio in the growth medium increased. Ammonium inhibited shoot and root growth; NO₃ alleviated the toxic effects of NH⁺₄. Soluble protein concentrations were higher in the shoots of NO₃-fed plants and in the roots of plants supplied with NH⁺₄. These results suggest that accumulation of A₂bu and other nonprotein amino acids, as well as asparagine and glutamine, plays a role in detoxification of NH⁺₄ and storage of N.     

Rubidium Uptake and Accumulation in Peripheral Myelinated Internodal Axons and Schwann Cells

Abstract: To study mechanisms of K⁺ transport in peripheral nerve, uptake of rubidium (Rb⁺), a K⁺ tracer, was characterized in rat tibial nerve myelinated axons and glia. Isolated nerve segments were perfused with zero-K⁺ Ringer's solutions containing Rb⁺ (1–20 m M ) and x-ray microanalysis was used to measure water content and concentrations of Rb, Na, K, and Cl in internodal axoplasm, mitochondria, and Schwann cell cytoplasm and myelin. Both axons and Schwann cells were capable of removing extracellular Rb⁺ (Rb⁺_o) and exchanging it for internal K⁺. Uptake into axoplasm, Schwann cytoplasm, and myelin was a saturable process over the 1–10 m M Rb⁺_o concentration range, although corresponding axoplasmic uptake rates were higher than respective glial velocities. Mitochondrial accumulation was a linear function of axoplasmic Rb⁺ concentrations, which suggests involvement of a nonenzymatic process. At 20 m M Rb⁺_o, a differential stimulatory response was observed; i.e., axoplasmic Rb⁺ uptake velocities increased more than fivefold relative to the 10 m M rate, and glial cytoplasmic uptake rose almost threefold. Finally, Rb⁺_o uptake rate into axons and glia was completely inhibited by ouabain (2–4 m M ) exposure or incubation at 4°C. These results suggest that Rb⁺ uptake into peripheral nerve internodal axons and Schwann cells is mediated by Na⁺,K⁺-ATPase activity and implicate the presence of axonal- and glial-specific Na⁺ pump isozymes.     

Characteristics of ammonium absorption by excised root and leaf tissues of maize

Absorption of ammonium from solutions of ammonium chloride by maize ( Zea mays L. cv. GS-2) tissue was studied. In contrast to an initial rapid phase of absorption in root tissue, a one hour lag period was recorded in leaf tissue. The maximum rate of uptake was observed at 5–10 m M NH₄Cl in both tissues. Roots had a K_m value of 1.0 m M and V_max of 24.3 μmol ammonium (g fresh weight)⁻¹ h⁻¹, whereas the leaf tissue had a higher K_m (4.1 m M ) and a lower V_max (8.7 μmol). There was a concentration dependent increase in ethanol soluble and insoluble fractions of organic nitrogen during ammonium supply. The optimum pH for ammonium absorption for both tissues was 7.4. The optimal concentration of CaCl₂ for ammonium absorption was 5 m M whereas that of KCl was only 1 m M . In both tissues, the absorption was inhibited substantially by DCMU, DNP, cycloheximide, lincomycin, sodium tungstate, sodium arsenate and to some extent also by the anions nitrate and sulfate. It is suggested that a carrier is involved in an active uptake of ammonium in the leaf tissues.     

Regulation of nitrate uptake into Chara corallina cells via NH+4 stimulation of NO−3 efflux

Abstract. Net NO₃ uptake by NO⁻₃ deficient Chara cells was used to calculate [NO⁻₃]_c assuming that the cytoplasm occupies 10% total volume and that nitrate reduction and storage are negligible (i.e. maximum [NO⁻₃]_c was calculated). A linear relationship was found between NO⁻₃ efflux and [NO⁻₃]_c. There was an initial burst of NO⁻₃ efflux when NH⁺₄ was added, followed by a slower efflux rate which matched influx rate such that net NO⁻₃ uptake was zero. Over 50% of NO⁻₃ that had been taken up in 2 h was lost within the first 5 min of NH⁺₄ addition. The Nernst equation was used to predict the direction of the electrochemical driving force for NO⁻₃ entry. Under the experimental conditions used NO⁻₃ efflux is actively transported. The differential involvement of both NO⁻₃ influx and NO⁻₃ efflux in the regulation of NO⁻₃ uptake is discussed and a model is proposed to account for these results which envisages discrete NO⁻₃ influx and NO⁻₃ efflux carriers.