In Vivo Blue-Light Activation of Chlamydomonas reinhardii Nitrate Reductase

Chlamydomonas reinhardii cells, growing photoautotrophically under air, excreted to the culture medium much higher amounts of NO₂⁻ and NH₄⁺ under blue than under red light. Under similar conditions, but with NO₂⁻ as the only nitrogen source, the cells consumed NO₂⁻ and excreted NH₄⁺ at similar rates under blue and red light. In the presence of NO₃⁻ and air with 2% CO₂ (v/v), no excretion of NO₂⁻ and NH₄⁺ occurred and, moreover, if the bubbling air of the cells that were currently excreting NO₂⁻ and NH₄⁺ was enriched with 2% CO₂ (v/v), the previously excreted reduced nitrogen ions were rapidly reassimilated. The levels of total nitrate reductase and active nitrate reductase increased several times in the blue-light-irradiated cells growing on NO₃⁻ under air. When tungstate replaced molybdate in the medium (conditions that do not allow the formation of functional nitrate reductase), blue light activated most of the preformed inactive enzyme of the cells. Furthermore, nitrate reductase extracted from the cells in its inactive form was readily activated in vitro by blue light. It appears that under high irradiance (90 w m⁻²) and low CO₂ tensions, cells growing on NO₃⁻ or NO₂⁻ may not have sufficient carbon skeletons to incorporate all the photogenerated NH₄⁺. Because these cells should have high levels of reducing power, they might use NO₃⁻ or, in its absence, NO₂⁻ as terminal electron acceptors. The excretion of the products of NO₂⁻ and NH₄⁺ to the medium may provide a mechanism to control reductant level in the cells. Blue light is suggested as an important regulatory factor of this photorespiratory consumption of NO₃⁻ and possibly of the whole nitrogen metabolism in green algae.     

Nitrate Reductase Activity in Shoots and Roots of Maize Seedlings as Affected by the Form of Nitrogen Nutrition and the pH of the Nutrient Solution

The effect of nitrogen form (NH₄-N, NH₄-N + NO₃⁻, NO₃⁻) on nitrate reductase activity in roots and shoots of maize (Zea mays L. cv INRA 508) seedlings was studied. Nitrate reductase activity in leaves was consistent with the well known fact that NO₃⁻ increases, and NH₄⁺ and amide-N decrease, nitrate reductase activity. Nitrate reductase activity in the roots, however, could not be explained by the root content of NO₃⁻, NH₄-N, and amide-N. In roots, nitrate reductase activity in vitro was correlated with the rate of nitrate reduction in vivo. Inasmuch as nitrate reduction results in the production of OH⁻ and stimulates the synthesis of organic anions, it was postulated that nitrate reductase activity of roots is stimulated by the released OH⁻ or by the synthesized organic anions rather than by nitrate itself. Addition of HCO₃⁻ to nutrient solution of maize seedlings resulted in a significant increase of the nitrate reductase activity in the roots. As HCO₃⁻, like OH⁻, increases pH and promotes the synthesis of organic anions, this provides circumstantial evidence that alkaline conditions and/or organic anions have a more direct impact on nitrate reductase activity than do NO₃⁻, NH₄-N, and amide-N.     

Spectral Dependence of Photoregulation of Inorganic Nitrogen Metabolism in Chlamydomonas reinhardii

The utilization of NO₃⁻ by green algae growing photoautotrophically under air, which are growth conditions close to their more habitual situations in nature, is associated with the excretion of NO₂⁻ and NH₄⁺ to the culture medium. The entire process is promoted by blue light and depends on photosynthetically active radiation for the required reducing equivalents. The stimulation of NO₃⁻ utilization and of its associated NO₂⁻ and NH₄⁺ excretions saturated at very low quantum fluxes of blue light (15 microequivalents per square meter per second) in Chlamydomonas reinhardii cells sparged with CO₂-free air and irradiated with 50 microequivalents per square meter per second background red light. The wavelength dependence data of this stimulation correlated closely with the in situ photoactivation of nitrate reductase and also with the light induced increase in its biosynthesis and/or assembly.

These results indicate that the photoregulation of inorganic N metabolism in C. reinhardii is mainly due to the blue light modulation of nitrate reductase. Although flavins are the most suitable candidates to act as physiological photoreceptors, the wavelength dependence data only show a major peak in the blue region between 400 and 500 nanometers.

     

Comparative studies on the induction and inactivation of nitrate reductase in corn roots and leaves

A comparison of induction and inactivation of nitrate reductase and two of its component activities, namely FMNH₂-nitrate reductase and NO₃⁻-induced NADH-cytochrome c reductase, was made in roots and leaves of corn (Zea mays L. var. W64A × 182E). The three activities were induced in parallel in both tissues when NO₃⁻ was supplied. WO₄⁼ suppressed the induction of NADH- and FMNH₂-nitrate reductase activities in root tips and leaves. The NO₃⁻-induced NADH-cytochrome c reductase activity showed a normal increase in roots treated with WO₄⁼. In leaves, on the other hand, there was a marked superinduction of the NO₃⁻-induced NADH-cytochrome c reductase in the presence of WO₄⁼.     

Enhancement of Nitrate Reductase Activity by Benzyladenine in Agrostemma githago

Nitrate reductase activity in excised embryos of Agrostemma githago increases in response to both NO₃⁻ and cytokinins. We asked the question whether cytokinins affected nitrate reductase activity directly or through NO₃⁻, either by amplifying the effect of low endogenous NO₃⁻ levels, or by making NO₃⁻ available for induction from a metabolically inactive compartment. Nitrate reductase activity was enhanced on the average by 50% after 1 hour of benzyladenine treatment. In some experiments, the cytokinin response was detectable as early as 30 minutes after addition of benzyladenine. Nitrate reductase activity increased linearly for 4 hours and began to decay 13 hours after start of the hormone treatment. When embryos were incubated in solutions containing mixtures of NO₃⁻ and benzyladenine, additive responses were obtained. The effects of NO₃⁻ and benzyladenine were counteracted by abscisic acid. The increase in nitrate reductase activity was inhibited at lower abscisic acid concentrations in embryos which were induced with NO₃⁻, as compared to embryos treated with benzyladenine. Casein hydrolysate inhibited the development of nitrate reductase activity. The response to NO₃⁻ was more susceptible to inhibition by casein hydrolysate than the response to the hormone. When NO₃⁻ and benzyladenine were withdrawn from the medium after maximal enhancement of nitrate reductase activity, the level of the enzyme decreased rapidly. Nitrate reductase activity increasd again as a result of a second treatment with benzyladenine but not with NO₃⁻. At the time of the second exposure to benzyladenine, no NO₃⁻ was detectable in extracts of Agrostemma embryos. This is taken as evidence that cytokinins enhance nitrate reductase activity directly and not through induction by NO₃⁻.     

Mechanism of Stimulation and Inhibition of Tonoplast H-ATPase of Beta vulgaris by Chloride and Nitrate

The H⁺-ATPase of tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue was studied with respect to the kinetic effects of Cl⁻ and NO₃⁻. N-Ethylmaleimide (NEM) was employed as a probe to investigate substrate binding and gross conformational changes of the enzyme. Chloride decreased the K_m of the enzyme for ATP but caused relatively little alteration of the V_max. Nitrate increased K_m only. Michaelis-Menten kinetics applied throughout with respect to ATP concentration. Nitrate yielded similar kinetics of inhibition in both the presence and absence of Cl⁻. Other monovalent anions that specifically increased the K_m of the ATPase for ATP were, in order of increasing K_i, SCN⁻, ClO₄⁻, and ClO₃⁻. Sulfate, although inhibitory, manifested noncompetitive kinetics with respect to ATP concentration. ADP, like NO₃⁻, was a competitive inhibitor of the ATPase but ADP and NO₃⁻ did not interact cooperatively nor did either interfere with the inhibitory action of the other. It is concluded that NO₃⁻ does not show competitive kinetics because of its stereochemical similarity to the terminal phosphoryl group of ATP. NEM was an irreversible inhibitor of the tonoplast ATPase. Both Mg·ADP and Mg·ATP protected the enzyme from inactivation by NEM but Mg·ADP was the more potent of the two. Chloride and NO₃⁻ exerted little or no effect on the protective actions of Mg·ADP and Mg·ATP suggesting that neither Cl⁻ nor NO₃⁻ are involved in substrate binding.     

In Vivo Determination of Parameters of Nitrate Utilization in Wheat (Triticum aestivum L.) Seedlings Grown with Low Concentration of Nitrate in the Nutrient Solution

Six genotypes of winter wheat (Triticum aestivum L.) differing in grain protein concentration were grown on a nutrient solution containing low concentrations of NO₃⁻ (2 millimolar). Total NO₃⁻ uptake varied between genotypes but was not related to grain protein content. An in vivo nitrate reductase assay was used to determine the affinity of the enzyme for NO₃⁻, and large phenotypic variations were observed. In vivo estimations of the concentration and size of the metabolic pool were variable. However, the three genotypes with the higher ratios of metabolic pool size to leaf total NO₃⁻ concentration were the high protein varieties. It is proposed that a high affinity of nitrate reductase for nitrate might be a biochemical marker for the capacity of the plant to continue assimilating NO₃⁻ for a longer period during the last stage of growth.     

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⁺.     

Differential light induction of nitrate reductases in greening and photobleached soybean seedlings

Soybean (Glycine max [L.] Merr.) seeds were imbibed and germinated with or without NO₃⁻, tungstate, and norflurazon (San 9789). Norflurazon is a herbicide which causes photobleaching of chlorophyll by inhibiting carotenoid synthesis and which impairs normal chloroplast development. After 3 days in the dark, seedlings were placed in white light to induce extractable nitrate reductase activity. The induction of maximal nitrate reductase activity in greening cotyledons did not require NO₃⁻ and was not inhibited by tungstate. Induction of nitrate reductase activity in norflurazon-treated cotyledons had an absolute requirement for NO₃⁻ and was completely inhibited by tungstate. Nitrate was not detected in seeds or seedlings which had not been treated with NO₃⁻. The optimum pH for cotyledon nitrate reductase activity from norflurazon-treated seedlings was at pH 7.5, and near that for root nitrate reductase activity, whereas the optimum pH for nitrate reductase activity from greening cotyledons was pH 6.5. Induction of root nitrate reductase activity was also inhibited by tungstate and was dependent on the presence of NO₃⁻, further indicating that the isoform of nitrate reductase induced in norflurazon-treated cotyledons is the same or similar to that found in roots. Nitrate reductases with and without a NO₃⁻ requirement for light induction appear to be present in developing leaves. In vivo kinetics (light induction and dark decay rates) and in vitro kinetics (Arrhenius energies of activation and NADH:NADPH specificities) of nitrate reductases with and without a NO₃⁻ requirement for induction were quite different. K_m values for NO₃⁻ were identical for both nitrate reductases.     

Denitrification and Assimilatory Nitrate Reduction in Aquaspirillum magnetotacticum

Aquaspirillum magnetotacticum MS-1 grew microaerobically but not anaerobically with NO₃⁻ or NH₄⁺ as the sole nitrogen source. Nevertheless, cell yields varied directly with NO₃⁻ concentration under microaerobic conditions. Products of NO₃⁻ reduction included NH₄⁺, N₂O, NO, and N₂. NO₂⁻ and NH₂OH, each toxic to cells at 0.2 mM, were not detected as products of cells growing on NO₃⁻. NO₃⁻ reduction to NH₄⁺ was completely repressed by the addition of 2 mM NH₄⁺ to the growth medium, whereas NO₃⁻ reduction to N₂O or to N₂ was not. C₂H₂ completely inhibited N₂O reduction to N₂ by growing cells. These results indicate that A. magnetotacticum is a microaerophilic denitrifier that is versatile in its nitrogen metabolism, concomitantly reducing NO₃⁻ by assimilatory and dissimilatory means. This bacterium appears to be the first described denitrifier with an absolute requirement for O₂. The process of NO₃⁻ reduction appears well adapted for avoiding accumulation of several nitrogenous intermediates that are toxic to cells.     

Root respiration associated with ammonium and nitrate absorption and assimilation by barley

We examined nitrate assimilation and root gas fluxes in a wild-type barley (Hordeum vulgare L. cv Steptoe), a mutant (nar1a) deficient in NADH nitrate reductase, and a mutant (nar1a;nar7w) deficient in both NADH and NAD(P)H nitrate reductases. Estimates of in vivo nitrate assimilation from excised roots and whole plants indicated that the nar1a mutation influences assimilation only in the shoot and that exposure to NO₃⁻ induced shoot nitrate reduction more slowly than root nitrate reduction in all three genotypes. When plants that had been deprived of nitrogen for several days were exposed to ammonium, root carbon dioxide evolution and oxygen consumption increased markedly, but respiratory quotient—the ratio of carbon dioxide evolved to oxygen consumed—did not change. A shift from ammonium to nitrate nutrition stimulated root carbon dioxide evolution slightly and inhibited oxygen consumption in the wild type and nar1a mutant, but had negligible effects on root gas fluxes in the nar1a;nar7w mutant. These results indicate that, under NH₄⁺ nutrition, 14% of root carbon catabolism is coupled to NH₄⁺ absorption and assimilation and that, under NO₃⁻ nutrition, 5% of root carbon catabolism is coupled to NO₃⁻ absorption, 15% to NO₃⁻ assimilation, and 3% to NH₄⁺ assimilation. The additional energy requirements of NO₃⁻ assimilation appear to diminish root mitochondrial electron transport. Thus, the energy requirements of NH₄⁺ and NO₃⁻ absorption and assimilation constitute a significant portion of root respiration.     

Short Term Studies of Nitrate Uptake into Barley Plants Using Ion-Specific Electrodes and ClO(3): II. Regulation of NO(3) Efflux by NH(4)

The influence of NH₄⁺, in the external medium, on fluxes of NO₃⁻ and K⁺ were investigated using barley (Hordeum vulgare cv Betzes) plants. NH₄⁺ was without effect on NO₃⁻ (³⁶ClO₃⁻) influx whereas inhibition of net uptake appeared to be a function of previous NO₃⁻ provision. Plants grown at 10 micromolar NO₃⁻ were sensitive to external NH₄⁺ when uptake was measured in 100 micromolar NO₃⁻. By contrast, NO₃⁻ uptake (from 100 micromolar NO₃⁻) by plants previously grown at this concentration was not reduced by NH₄⁺ treatment. Plants pretreated for 2 days with 5 millimolar NO₃⁻ showed net efflux of NO₃⁻ when roots were transferred to 100 micromolar NO₃⁻. This efflux was stimulated in the presence of NH₄⁺. NH₄⁺ also stimulated NO₃⁻ efflux from plants pretreated with relatively low nitrate concentrations. It is proposed that short term effects on net uptake of NO₃⁻ occur via effects upon efflux. By contrast to the situation for NO₃⁻, net K⁺ uptake and influx of ³⁶Rb⁺-labeled K⁺ was inhibited by NH₄⁺ regardless of the nutrient history of the plants. Inhibition of net K⁺ uptake reached its maximum value within 2 minutes of NH₄⁺ addition. It is concluded that the latter ion exerts a direct effect upon K⁺ influx.     

Hydroxylamine inhibition of the nitrate reductase complex from Amaranthus

Nitrate reductase from Amaranthus viridis is similar to nitrate reductase from other plant sources. NH₂OH inhibits nitrate reduction from NADH by the nitrate reductase complex, but it does not inhibit either the NADH-dehydrogenase activity or nitrate reduction from reduced flavin mononucleotides. The inhibition observed was non-competitive with nitrate when the enzyme was pre-incubated with NH₂OH and NADH, and competitive with nitrate without pre-incubation. The K_i values for NH₂OH were 5 μM and 30 μM with or without pre-incubation respectively.     

Nodule and Leaf Nitrate Reductases and Nitrogen Fixation in Medicago sativa L. under Water Stress

The effect of water stress on patterns of nitrate reductase activity in the leaves and nodules and on nitrogen fixation were investigated in Medicago sativa L. plants watered 1 week before drought with or without NO₃⁻. Nitrogen fixation was decreased by water stress and also inhibited strongly by the presence of NO₃⁻. During drought, leaf nitrate reductase activity (NRA) decreased significantly particularly in plants watered with NO₃⁻, while with rewatering, leaf NRA recovery was quite important especially in the NO₃⁻-watered plants. As water stress progressed, the nodular NRA increased both in plants watered with NO₃⁻ and in those without NO₃⁻ contrary to the behavior of the leaves. Beyond −15.10⁵ pascal, nodular NRA began to decrease in plants watered with NO₃⁻. This phenomenon was not observed in nodules of plants given water only.     

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.     

Aspects of nitrogen metabolism in the rice seedling

The effects of nitrogen source NO₃⁻ or NH₄⁺ on nitrogen metabolism during the first 2 weeks of germination of the rice seedling (Oryza sativa L., var. IR22) grown in nutrient solution containing 40 μg/ml N were studied. Total, soluble protein, and free amino N levels were higher in the NH₄⁺-grown seedling, particularly during the 1st week of germination. Asparagine accounted for most of the difference in free amino acid level, in both the root and the shoot. Nitrate and nitrite reductase activities were present mainly in the shoot and were higher in the NO₃⁻-grown seedling, whereas the activity of glutamate dehydrogenase and glutamine synthetase in the root tended to be lower than that of the NH₄⁺-grown seedling during the 1st week of germination. Glycolate oxidase and catalase activities were present mainly in the shoot. Maximum activity of the above five enzymes occurred 7 to 10 days after germination. Differences in the zymograms of nitrate reductase, glutamate dehydrogenase, and catalase were mainly between shoot and root and not from N source. Nitrite reductase bands were observed only in plants grown in plants grown in NO₃⁻.     

Nitrate Reductase Activity in Soybeans (Glycine max [L.] Merr.): II. Energy Limitations

Growth chamber studies with soybeans (Glycine max [L.] Merr.) were designed to determine the relative limitations of NO₃⁻, NADH, and nitrate reductase (NR) per se on nitrate metabolism as affected by light and temperature. Three NR enzyme assays (+NO₃⁻in vivo, −NO₃⁻in vivo, and in vitro) were compared. NR activity decreased with all assays when plants were exposed to dark. Addition of NO₃⁻ to the in vivo NR assay medium increased activity (over that of the −NO₃⁻in vivo assay) at all sampling periods of a normal day-night sequence (14 hr-30 C day; 10 hr-20 C night), indicating that NO₃⁻ was rate-limiting. The stimulation of in vivo NR activity by NO₃⁻ was not seen in plants exposed to extended dark periods at elevated temperatures (16 hr-30 C), indicating that under those conditions, NO₃⁻ was not the limiting factor. Under the latter condition, in vitro NR activity was appreciable (19 μmol NO₂⁻ [g fresh weight, hr]⁻¹) suggesting that enzyme level per se was not the limiting factor and that reductant energy might be limiting.     

Nitrate-Dependent Ferrous Iron Oxidation by Anaerobic Ammonium Oxidation (Anammox) Bacteria

We examined nitrate-dependent Fe²⁺ oxidation mediated by anaerobic ammonium oxidation (anammox) bacteria. Enrichment cultures of “Candidatus Brocadia sinica” anaerobically oxidized Fe²⁺ and reduced NO₃⁻ to nitrogen gas at rates of 3.7 ± 0.2 and 1.3 ± 0.1 (mean ± standard deviation [SD]) nmol mg protein⁻¹ min⁻¹, respectively (37°C and pH 7.3). This nitrate reduction rate is an order of magnitude lower than the anammox activity of “Ca. Brocadia sinica” (10 to 75 nmol NH₄⁺ mg protein⁻¹ min⁻¹). A ¹⁵N tracer experiment demonstrated that coupling of nitrate-dependent Fe²⁺ oxidation and the anammox reaction was responsible for producing nitrogen gas from NO₃⁻ by “Ca. Brocadia sinica.” The activities of nitrate-dependent Fe²⁺ oxidation were dependent on temperature and pH, and the highest activities were seen at temperatures of 30 to 45°C and pHs ranging from 5.9 to 9.8. The mean half-saturation constant for NO₃⁻ ± SD of “Ca. Brocadia sinica” was determined to be 51 ± 21 μM. Nitrate-dependent Fe²⁺ oxidation was further demonstrated by another anammox bacterium, “Candidatus Scalindua sp.,” whose rates of Fe²⁺ oxidation and NO₃⁻ reduction were 4.7 ± 0.59 and 1.45 ± 0.05 nmol mg protein⁻¹ min⁻¹, respectively (20°C and pH 7.3). Co-occurrence of nitrate-dependent Fe²⁺ oxidation and the anammox reaction decreased the molar ratios of consumed NO₂⁻ to consumed NH₄⁺ (ΔNO₂⁻/ΔNH₄⁺) and produced NO₃⁻ to consumed NH₄⁺ (ΔNO₃⁻/ΔNH₄⁺). These reactions are preferable to the application of anammox processes for wastewater treatment.     

Early effects of salinity on nitrate assimilation in barley seedlings

The effect of NaCl and Na₂SO₄ salinity on NO₃⁻ assimilation in young barley (Hordeum vulgare L. var Numar) seedlings was studied. The induction of the NO₃⁻ transporter was affected very little; the major effect of the salts was on its activity. Both Cl⁻ and SO₄²⁻ salts severely inhibited uptake of NO₃⁻. When compared on the basis of osmolality of the uptake solutions, Cl⁻ salts were more inhibitory (15-30%) than SO₄²⁻ salts. At equal concentrations, SO₄²⁻ salts inhibited NO₃⁻ uptake 30 to 40% more than did Cl⁻ salts. The absolute concentrations of each ion seemed more important as inhibitors of NO₃⁻ uptake than did the osmolality of the uptake solutions. Both K⁺ and Na⁺ salts inhibited NO₃⁻ uptake similarly; hence, the process seemed more sensitive to anionic salinity than to cationic salinity.

Unlike NO₃⁻ uptake, NO₃⁻ reduction was not affected by salinity in short-term studies (12 hours). The rate of reduction of endogenous NO₃⁻ in leaves of seedlings grown on NaCl for 8 days decreased only 25%. Nitrate reductase activity in the salt-treated leaves also decreased 20% but its activity, determined either in vitro or by the `anaerobic' in vivo assay, was always greater than the actual in situ rate of NO₃⁻ reduction. When salts were added to the assay medium, the in vitro enzymic activity was severely inhibited; whereas the anaerobic in vivo nitrate reductase activity was affected only slightly. These results indicate that in situ nitrate reductase activity is protected from salt injury. The susceptibility to injury of the NO₃⁻ transporter, rather than that of the NO₃⁻ reduction system, may be a critical factor to plant survival during salt stress.

     

Influence of Nitrate and Ammonium Nutrition on the Uptake, Assimilation, and Distribution of Nutrients in Ricinus communis

Ricinus communis L. plants were grown in nutrient solutions in which N was supplied as NO₃⁻ or NH₄⁺, the solutions being maintained at pH 5.5. In NO₃⁻-fed plants excess nutrient anion over cation uptake was equivalent to net OH⁻ efflux, and the total charge from NO₃⁻ and SO₄²⁻ reduction equated to the sum of organic anion accumulation plus net OH⁻ efflux. In NH₄⁺-fed plants a large H⁺ efflux was recorded in close agreement with excess cation over anion uptake. This H⁺ efflux equated to the sum of net cation (NH₄⁺ minus SO₄²⁻) assimilation plus organic anion accumulation. In vivo nitrate reductase assays revealed that the roots may have the capacity to reduce just under half of the total NO₃⁻ that is taken up and reduced in NO₃⁻-fed plants. Organic anion concentration in these plants was much higher in the shoots than in the roots. In NH₄⁺-fed plants absorbed NH₄⁺ was almost exclusively assimilated in the roots. These plants were considerably lower in organic anions than NO₃⁻-fed plants, but had equal concentrations in shoots and roots. Xylem and phloem saps were collected from plants exposed to both N sources and analyzed for all major contributing ionic and nitrogenous compounds. The results obtained were used to assist in interpreting the ion uptake, assimilation, and accumulation data in terms of shoot/root pH regulation and cycling of nutrients.