Nitrate Utilization by the Diatom Skeletonema costatum: II. Regulation of Nitrate Uptake

Nitrate utilization has been characterized in nitrogen-deficient cells of the marine diatom Skeletonema costatum. In order to separate nitrate uptake from nitrate reduction, nitrate reductase activity was suppressed with tungstate. Neither nitrite nor the presence of amino acids in the external medium or darkness affects nitrate uptake kinetics. Ammonium strongly inhibits carrier-mediated nitrate uptake, without affecting diffusion transfer. A model is proposed for the uptake and assimilation of nitrate in S. costatum and their regulation by ammonium ions.     

The Influence of Ambient Nitrate, Temperature, and Light on Nitrate Assimilation in Sudangrass Seedlings

Seedlings of Sundangrass (Sorghum Sudanese [Piper] Stapf.) were grown 10 to 13 days of age in a nutrient solution containing nitrate and then placed under treatment conditions for 24 h before assays of nitrate assimilation were begun. Nitrate uptake was determined by its disappearance from the ambient solution. In vivo reduction of nitrate was determined by the overall balance between the amount taken up and the change in tissue concentration of nitrate during the experiments. Nitrate reductase activity was determined from tissue slices. In vivo reduction was strongly regulated by uptake in response to time and ambient nitrate concentration, temperature and light. Nitrate reduction responded to the concentration of nitrate supplied by uptake and by a storage pool, since reduction often exceeded uptake. Nitrate reductase activity in tissue slices was exponential in initial response to increasing temperature. After a 24-h equilibration period at each temperature, the activity was lower at higher temperatures. In contrast, actual reduction of nitrate increased linearly with increasing temperature between 15 and 24°C in the plants equilibrated 24 h at each temperature. Nitrate uptake and reduction were greatly inhibited under low light conditions, with reduction inhibited more than uptake., The effect of ambient nitrate, temperature, and light on the nitrate assimilatory processes help to explain observations reported on nitrate accumulation by Sudangrass forage.     

Trichlorfon-Induced Inhibition of Nitrate and Ammonium Uptake in Cyanobacteria

The possibility that the primary effect of the toxic insecticidetrichlorfon is an inhibition of nitrate uptake in cyanobactenahas been investigated. A drastic reduction in the rate of uptakeis detected 3 h after the addition of the insecticide to batchcultures of nabaena PCC 7119. The dose-response curves indicatea relationship between the degree of inhibition of nitrate uptakeand the reduction of chlorophyll content and growth. Nitratereductase (ferredoxin : nitrate reductase, EC 1.7.99.4 [EC] ) activityis also lowered as a result of insecticide action. When AnabaenaPCC 7119 cells are grown with ammonium as a source of combinednitrogen, trichlorfon reduces the rate of ammonium uptake. Therate of uptake of both nitrate and ammonium is restored uponwashing the cells. Ultrastructural analysis of Anabaena nitrate-growncells shows that trichlorfon does not damage thylakoid membranes,but brings about the accumulation of enlarged cyanophycin granulesand the increase of carboxysome number. Nitrate uptake rateand chlorophyll and phycobiliprotein contents are also reducedby insecticide treatment in the cyanobacteria SynechococcusUAM 211, GloeothecePCC 6501, Plectonema calothricoides, NostocUAM 205 and Chlorogloeopsis PCC 6912. These results are consistentwith the inhibition of nitrate uptake due to weak adsorptionof trichlorfon to the plasmalemma being the main effect of theinsecticide on cyanobacterial metabolism. Key words: Nitrate uptake, cyanobacteria, Anabaena, ammonium uptake, trichlorfon     

Nitrate reductase activity as a function of in situ nitrate uptake and environmental factors of euphotic zone profiles

The activity of nitrate reductase in euphotic zone profiles from several oceanic areas has been compared with in situ nitrate uptake rates estimated by the ¹⁵N technique. There were marked variations in the ratio of uptake to reduction and these have been related to the available light intensity, nitrate concentration, and the absolute value of nitrate uptake. It is shown that uptakereduction ratios greater than one are not due to dephasing between the two processes and that accumulation of nitrate inside the cells cannot account for the discrepancy. Nitrate uptake depends primarily on the external nitrate concentration, while nitrate reductase activity seems to be controlled by the intracellular nitrate level. The importance of those results as regards the significance of the nitrate reductase assay as an index of nitrate assimilation by marine phytoplankton is discussed.     

Nitrate transport system in Neurospora crassa

Nitrate uptake in Neurospora crassa has been investigated under various conditions of nitrogen nutrition by measuring the rate of disappearance of nitrate from the medium and by determining mycelial nitrate accumulation. The nitrate transport system is induced by either nitrate or nitrite, but is not present in mycelia grown on ammonia or Casamino Acids. The appearance of nitrate uptake activity is prevented by cycloheximide, puromycin, or 6-methyl purine. The induced nitrate transport system displays a K_m for nitrate of 0.25 mM. Nitrate uptake is inhibited by metabolic poisons such as 2,4-dinitrophenol, cyanide, and antimycin A. Furthermore, mycelia can concentrate nitrate 50-fold. Ammonia and nitrite are non-competitive inhibitors with respect to nitrate, with K_i values of 0.13 and 0.17 mM, respectively. Ammonia does not repress the formation of the nitrate transport system. In contrast, the nitrate uptake system is repressed by Casamino Acids. All amino acids individually prevent nitrate accumulation, with the exception of methionine, glutamine, and alanine. The influence of nitrate reduction and the nitrate reductase protein on nitrate transport was investigated in wild-type Neurospora lacking a functional nitrate reductase and in nitrate non-utilizing mutants, nit-1, nit-2, and nit-3. These mycelia contain an inducible nitrate transport system which displays the same characteristics as those found in the wild-type mycelia having the functional nitrate reductase. These findings suggest that nitrate transport is not dependent upon nitrate reduction and that these two processes are separate events in the assimilation of nitrate.     

The Apparent Induction of Nitrate Uptake by Chara corallina Cells following Pretreatment with or without Nitrate and Chlorate

Nitrate provision has been found to regulate the capacity forChara corallina cells to take up nitrate. When nitrate was suppliedto N sufficient cells maximum nitrate uptake was reached after8 h. Prolonged treatment of the cells in the absence of N alsoresulted in the apparent ability of these cells to take up nitrate.Chlorate was found to substitute partially for nitrate in the‘induction’ step. The effects on nitrate reductionwere separated from those on nitrate uptake by experiments usingtungstate. Tungstate pretreatment had no effect on NO^–₃uptake ‘induced’ by N starvation, but inhibitedNO^–₃ uptake associated with NO^–₃ pretreatment. Chloridepretreatment similarly had no effect on NO^–₃ uptake ‘induced’by N deprivation, but inhibited NO^–₃ uptake followingNO^–₃ pretreatment. The data suggest that there are atleast two mechanisms responsible for the ‘induction’of nitrate uptake by Chara cells, one associated with NO^–₃reduction and ‘induced’ by CIO^–₃ or NO^–₃and one associated with N deprivation. Key words: Nitrate, Chlorate, Chara corallina, Induction     

Nitrate transport in Zea mays cell suspension cultures: the effect of solution composition and cell age

Nitrate transport characteristics of an amino acid-grown Zeamays P3377 cell culture line were studied. Age (days after subculturing)of the cells was shown to have a significant effect on transport; older stationary phasecultures did not absorb nitrate from the medium as rapidly asyounger growing cultures. Solution composition had a pronouncedimpact on induction of accelerated nitrate transport and transportrates. Maximum uptake rates required fresh culture media ratherthan simple solutions. Differences in ionic strength among uptakesolutions of equal concentration were shown to affect the apparent uptake rates by changing theactivity coefficient of . The uptake kinetics were established by following uptake for 24h in a wide range of nitrate concentrations. Uptake patternsof cells in solutions ranging from 0.02 to 2 mM were as typicallyreported for plants. The kinetic constants for the Zea mayscell suspension cultures concurred with reports of other solution-culturedcells. When cells were placed in solutions containing greater than 2 mM, uptake patternssuggested a significant passive uptake component. Passive diffusionof was estimated by Nernst analysis and indicated to be an important component of nitrateuptake in maize cell suspension cultures grown in the absenceof nitrate then transferred into nitrate-containing media. Key words: Cell suspension culture, nitrate, passive uptake, Zea mays     

Nitrate enhances the survival of Mycobacterium tuberculosis during inhibition of respiration

When oxygen is slowly depleted from growing cultures of Mycobacterium tuberculosis, they enter a state of nonreplicating persistence that resembles the dormant state seen with latent tuberculosis. In this hypoxic state, nitrate reductase activity is strongly induced. Nitrate in the medium had no effect on long-term persistence during gradual oxygen depletion (Wayne model) for up to 46 days, but significantly enhanced survival during sudden anaerobiosis. This enhancement required a functional nitrate reductase. Thioridazine is a member of the class of phenothiazines that act, in part, by inhibiting respiration. Thioridazine was toxic to both actively growing and nonreplicating cultures of M. tuberculosis. At a sublethal concentration of thioridazine, nitrate in the medium improved the growth. At lethal concentrations of thioridazine, nitrate increased survival during aerobic incubation as well as in microaerobic cultures that had just entered nonreplicating persistence (NRP-1). In contrast, the survival of anaerobic persistent (NRP-2) cultures exposed to thioridazine was not increased by the addition of nitrate. Nitrate reduction is proposed to play a role during the sudden interruption of aerobic respiration due to causes such as hypoxia, thioridazine, or nitric oxide.     

Uptake of Nitrate by Mutants of Arabidopsis thaliana, Disturbed in Uptake or Reduction of Nitrate

A study of nitrate and chlorate uptake by Arabidopsis thaliana was made with a wildtype and two mutant types, both mutants having been selected by resistance to high chlorate concentrations. All plants were grown on a nutrient solution with nitrate and/or ammonium as the nitrogen source. Uptake was determined from depletion in the ambient solution. Nitrate and chlorate were able to induce their own uptake mechanisms. Plants grown on ammonium nitrate showed a higher subsequent uptake rate of nitrate and chlorate than plants grown on ammonium alone. Mutant B25, which has no nitrate reductase activity, showed higher rates of nitrate and chlorate uptake than the wildtype, when both types were grown on ammonium nitrate. Therefore, the uptake of nitrate is not dependent on the presence of nitrate reductase. Nitrate has a stimulating effect on nitrate and chlorate uptake, whereas some product of nitrate and ammonium assimilation inhibits uptake of both ions by negative feedback. Mutant B 1, which was supposed to have a low chlorate uptake rate, also has disturbed uptake characteristics for nitrate.     

Uptake of Nitrate by the Diatom Phaeodactylum tricornutum

Ammonium-grown cells of P. tricornutum lack ability to takeup nitrate. This ability develops during 3 h of nitrogen-deprivation;development requires concurrent photosynthesis, and is inhibitedby cycloheximide. Nitrate is accumulated by cells with a developednitrate uptake mechanism. Nitrate uptake is inhibited by 10^–5M CCCP and, in darkness, by anaerobiosis; it therefore presumablyrequires ATP which can be supplied by either photophosphorylationor oxidative phosphorylation.     

Nitrate Transport Is Independent of NADH and NAD(P)H Nitrate Reductases in Barley Seedlings

Barley (Hordeum vulgare L.) has NADH-specific and NAD(P)H-bispecific nitrate reductase isozymes. Four isogenic lines with different nitrate reductase isozyme combinations were used to determine the role of NADH and NAD(P)H nitrate reductases on nitrate transport and assimilation in barley seedlings. Both nitrate reductase isozymes were induced by nitrate and were required for maximum nitrate assimilation in barley seedlings. Genotypes lacking the NADH isozyme (Az12) or the NAD(P)H isozyme (Az70) assimilated 65 or 85%, respectively, as much nitrate as the wild type. Nitrate assimilation by genotype (Az12;Az70) which is deficient in both nitrate reductases, was only 13% of the wild type indicating that the NADH and NAD(P)H nitrate reductase isozymes are responsible for most of the nitrate reduction in barley seedlings. For all genotypes, nitrate assimilation rates in the dark were about 55% of the rates in light. Hypotheses that nitrate reductase has direct or indirect roles in nitrate uptake were not supported by this study. Induction of nitrate transporters and the kinetics of net nitrate uptake were the same for all four genotypes indicating that neither nitrate reductase isozyme has a direct role in nitrate uptake in barley seedlings.     

Nitrate translocation by detopped corn seedlings

Five-day-old seedlings of corn (Zealpha mays L.) grown without nitrate were decapitated and exposed to 0.5 mm KNO(3) or 0.5 mm KCl in aerated solutions at 30 C. Uptake of nitrate, chloride, and potassium was determined by replacing solutions hourly and measuring their depletion. Translocation of these ions and of organic nitrogen was determined by hourly analysis of the vascular exudate. Nitrate reduction was estimated by the difference between nitrate uptake and nitrate recovered in the tissue and exudate. Nitrate uptake exhibited its usual pattern of apparent induction resulting in the development of an accelerated uptake phase. Chloride uptake remained fairly constant throughout the experimental period. Translocation of nitrate increased progressively for at least 7 hours whereas chloride translocation reached a maximum about the 3d hour and then declined to a lower rate than nitrate translocation. Nitrate uptake and translocation were restricted by anaerobiosis, by 20 and 40 C relative to 30 C, and by 0.05 mm 6-methylpurine, an RNA-synthesis inhibitor. Accumulation, reduction and translocation of nitrate had different sensitivities to all these factors. The effect of 0.05 mm 6-methylpurine was more detrimental to nitrate translocation and nitrate reduction than to nitrate uptake.Ambient nitrate, relative to chloride, enhanced the exudation volume and the translocation of organic nitrogen within 4 hours from initiation of the experiments. Translocation of nitrate and organic nitrogen decreased shortly after removal of external nitrate. The higher rates of organic nitrogen translocation which occurred during nitrate uptake indicates either (a) rapid translocation of amino acids synthesized from the entering nitrate, or (b) an accelerated rate of protein turnover and a resulting enhancement in translocation of endogenous amino acids.     

Regulation of Nitrate Uptake in Penicillium chrysogenum by Ammonium Ion

A nitrate uptake system is induced (along with nitrate reductase) when NH₄⁺-grown Penicillium chrysogenum is incubated with inorganic nitrate in synthetic medium in the absence of NH₄⁺. Nitrate uptake and nitrate reduction are probably in steady state in fully induced mycelium, but the ratios of the two activities are not constant during the induction period. Substrate concentrations of ammonium cause a rapid decay of nitrate uptake and nitrate reductase activity. The two activities are differentially inactivated (the uptake activity being more sensitive). Glutamine and asparagine are as effective as NH₄⁺ in suppressing nitrate uptake activity. Glutamate and alanine were about half as effective as NH₄⁺. Cycloheximide interferes with the NH₄⁺-induced decay of nitrate uptake activity. The ammonium transport system is almost maximally deinhibited (or derepressed) in nitrate-grown mycelium.     

Nitrate Uptake by Reef Corals

In specimens of the hermatypic coral species Fungia scutaria and Montipora verrucosa and in the alga Ulva lactuca, nitrate uptake was measured in light and dark with a flow-through apparatus. The nitrate uptake was measurable in high-nitrate bay water of Kaneohe Bay and also in low-nitrate open ocean water. Nitrate consumption rates by the corals and the alga did not differ from light to dark. Neither the coral nor the alga showed measurable immediate nitrate uptake in open ocean water of low nitrate concentration when they had been held previously in the high-nitrate bay water. In low-nitrate open ocean water the uptake per unit time increases when the flow of the water increases. The uptake of nitrate by reef corals even from low concentrations indicates nonspecific nutrient sources for reef corals.     

Evidence for multiple nitrate uptake systems in the yeast Hansenula polymorpha

Hansenula polymorpha mutants disrupted in the high-affinity nitrate transporter gene (YNT1) are still able to grow in nitrate. To detect the nitrate transporter(s) responsible for this growth a strain containing disruption of the nitrate assimilation gene cluster and expressing nitrate reductase gene (YNR1) under the control of H. polymorpha MOX1 (methanol oxidase) promoter was used (FM31 strain). In this strain nitrate taken up is transformed into nitrite by nitrate reductase and excreted to the medium where it is easily detected. Nitrate uptake which is neither induced by nitrate nor repressed by reduced nitrogen sources was detected in the FM31 strain. Likewise, nitrate uptake detected in the strain FM31 is independent of both Ynt1p and Yna1p and is not affected by ammonium, glutamine or chlorate. The inhibition of nitrite extrusion by extracellular nitrite suggests that the nitrate uptake system shown in the FM31 strain could also be involved in nitrite uptake.     

Nitrate transport and its regulation by O2 in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an obligate respirer which can utilize nitrate as a terminal electron acceptor under anaerobic conditions (denitrification). Immediate, transient regulation of nitrate respiration is mediated by oxygen through the inhibition of nitrate uptake. In order to gain an understanding of the bioenergetics of nitrate transport and its regulation by oxygen, the effects of various metabolic inhibitors on the uptake process and on oxygen regulation were investigated. Nitrate uptake was stimulated by the protonophores carbonyl cyanide m-chlorophenylhydrazone and 2,4-dinitrophenol, indicating that nitrate uptake is not strictly energized by, but may be affected by the proton motive force. Oxygen regulation of nitrate uptake might in part be through redox-sensitive thiol groups since N-ethylmaleimide at high concentrations decreased the rate of nitrate transport. Cells grown with tungstate (deficient in nitrate reductase activity) and azide-treated cells transported nitrate at significantly lower rates than untreated cells, indicating that physiological rates of nitrate transport are dependent on nitrate reduction. Furthermore, tungstate grown cells transported nitrate only in the presence of nitrite, lending support to the nitrate/nitrite antiport model for transport. Oxygen regulation of nitrate transport was relieved (10% that of typical anaerobic rates) by the cytochrome oxygen reductase inhibitors carbon monoxide and cyanide.     

Nitrate reductase activity and uptake of nitrate and oxygen as affected by nitrate supply to detached maize organs

Supply of 1, 2, 5, 10 or 20 mM nitrate to detached roots, scutella or shoots from 5- to 6-d-old Zea mays L. seedlings increased in vitro nitrate reductase (NR) activity in all the organs and NADPH specific NR (NADPH:NR) activity in roots and scutella but not in the shoots. Usually 2 to 5 mM nitrate supported maximum enzyme activity, the higher concentration did not increase it further. The protein content in the roots, scutella and shoots increased up to 5, 2 and 20 mM medium nitrate, respectively. Nitrate uptake also increased with increasing nitrate concentration in roots and shoots, but it increased only slightly in the scutella. In both roots and scutella, methionine sulfoximine had no effect, while cycloheximide and tungstate abolished nitrate induced NADH:NR activity completely and NADPH:NR partially. Methionine sulfoximine increased nitrate uptake by roots and scutella slightly, but other inhibitors had no effect. The depletion of dissolved oxygen from the medium was lower in the presence of nitrate than in its absence or in the presence of ammonium, especially in the scutella. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nitrate reduction and assimilation in Chlorella

1. Nitrate reduction and assimilation have been studied in Chlorella pyrenoidosa under growth conditions by observing effects on the CO(2)/O(2) gas exchange quotient. 2. During assimilation of glucose in the dark, nitrate reduction is noted as an increase in the R.Q. to about 1.6 caused by an increased rate of carbon dioxide production. 3. During photosynthesis at low light intensity nitrate reduction is evidenced by a reduction in the CO(2)O(2) quotient to about 0.7 caused by a decreased rate of carbon dioxide uptake. 4. Chlorella will assimilate nitrogen from either nitrate or ammonia. When both sources are supplied, only ammonia is utilized and no nitrate reduction occurs. It is inferred that under the usual conditions of growth nitrate is reduced only at a rate required for subsequent cellular syntheses. The effect of nitrate reduction on the CO(2)O(2) quotient therefore provides a measure of the relative rate of nitrogen assimilation. 5. Over-all photosynthetic metabolism may be described from elementary analysis of the cells since excretory products are negligible. The gas exchange predicted in this way is in good agreement with the observed CO(2)/O(2) quotients.     

Integrated Control of Nitrate Uptake by Crop Growth Rate and Soil Nitrate Availability under Field Conditions

There is still disagreement about whether crop growth rate orsoil nitrate concentration control nitrogen absorption by cropsunder field conditions. The influence of these factors on thecontrol of N uptake rate was examined in the absence of waterstress, using data on dry matter production, above-ground nitrogenaccumulation and soil nitrate concentration from several N-fertilizerexperiments on winter wheat, winter oilseed rape and maize.The results confirmed that crops can accumulate nitrogen farin excess of the ‘critical dilution curve’, whichdefines the minimum amount of nitrogen needed for maximal growthrate: the N concentration in plants could exceed the criticalN concentration by 70 to 80% for the three species studied.The nitrate uptake rate index (NUI) was calculated as the ratioof actual and critical N uptake rates, at intervals of 1 week.NUI varied with nitrate concentration in the 0–30 cm soillayer according to a Michaelis–Menten equation (with oneor two components). This response was compared with the kineticsof saturation of the nitrate uptake systems: the high affinitytransport system (HATS) and the low affinity transport system(LATS). As a result, it is proposed that there is a criticalN dilution curve delimiting two domains of N use by plants.This is linked to the two nitrate transport systems, with HATSworking at low nitrate concentrations, below the critical dilutioncurve, and LATS at high nitrate concentrations, above the curve.NUI provides another method for calculating the actual nitrateuptake rate, which depends on the maximal crop growth rate (withoutN deficiency) and on the external nitrate concentration. Copyright2000 Annals of Botany Company Nitrate, uptake rate, growth rate, wheat, maize, oilseed rape, soil N availability     

Nitrate uptake in the halotolerant cyanobacterium Aphanothece halophytica is energy-dependent driven by DeltapH

The energetics of nitrate uptake by intact cells of the halotolerant cyanobacterium Aphanothece halophytica were investigated. Nitrate uptake was inhibited by various protonophores suggesting the coupling of nitrate uptake to the proton motive force. An artificially-generated pH gradient across the membrane (DeltapH) caused an increase of nitrate uptake. In contrast, the suppression of DeltapH resulted in a decrease of nitrate uptake. The increase of external pH also resulted in an enhancement of nitrate uptake. The generation of the electrical potential across the membrane (Deltapsi) resulted in no elevation of the rate of nitrate uptake. On the other hand, the valinomycin-mediated dissipation of Deltapsi caused no depression of the rate of nitrate uptake. Thus, it is unlikely that Deltapsi participated in the energization of the uptake of nitrate. However, Na(+)-gradient across the membrane was suggested to play a role in nitrate uptake since monensin which collapses Na(+)-gradient strongly inhibited nitrate uptake. Exogenously added glucose and lactate stimulated nitrate uptake in the starved cells. N, N'-dicyclohexylcarbodiimide, an inhibitor of ATPase, could alsoinhibit nitrate uptake suggesting that ATP hydrolysis was required for nitrate uptake. All these results indicate that nitrate uptake in A. halophytica is ATP-dependent, driven by DeltapH and Na(+)-gradient.