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.     

Repression of nitrate reductase in cucumber leaves caused by calcium deficiency

Growth of young cucumber plants was strongly inhibited, whencalcium was removed from the culture solution. The activitiesof nitrate reductase, glutamate dehydrogenase and glutaminesynthetase were investigated after the removal of calcium. Thoughthe activities of glutamine synthetase and glutamate dehydrogenasewere not altered much, nitrate reductase activity, measuredby in vitro and in vivo assays, decreased dramatically. Theloss of nitrate reductase activity coincided with the levelof nitrate in the leaves. When nitrate was supplied to the cucumberswith a nitrate deficiency, the plants induced nitrate reductasetogether with a distinct accumulation of nitrate. However, cucumberstreated for both calcium and nitrate deficiency failed to inducenitrate reductase and to accumulate nitrate on the additionof large amounts of nitrate. Leaf sections that had been treatedfor both calcium and nitrate deficiency could induce nitratereductase when floated on nitrate solution under the light.This indicates that the drastic loss of nitrate reductase causedby the removal of calcium was due mainly to the deficiency ofnitrate as the inducer in leaves. (Received December 19, 1979; )     

Nitrate utilization in barley: relations to nitrate supply and light/dark cycles

Uptake and utilization of nitrate were investigated in Hordeum vulgare L. cvs Mette and Golf in the vegetative stage, 2 and 4 weeks after sowing. The plants were subjected to a light/dark cycle of 16/8 h (18/12°C). Results obtained with the two genotypes were essentially similar. In the light, xylem nitrate transport and shoot nitrate reduction approximately equalled the amount of nitrate absorbed by the root. A drastic decline in translocation to the shoot in darkness was entirely attributable to decreased transpiration since no major changes in xylem nitrate concentration were observed. Darkening caused only a slight decrease in nitrate uptake, while root nitrate reduction was enhanced. Nitrate starvation for 2 days did not significantlly affect dry matter increment, but resulted in a drastic drop in previously accumulated nitrate, indicating that the stored nitrate is accessible and can sustain unrestricted growth. Uptake increased upon re-addition of nitrate and after 8 h it was about twice that of non-starved plants. During recovery, restoration of root nitrate pools and root nitrate reduction took precedence over shoot nitrate accumulation and reduction. Net nitrate uptake and removal of nitrate from the root to the transpiration stream seem to be decisive for the rate of root nitrate reduction.     

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.     

Blood plasma response and urinary excretion of nitrite and nitrate in milk-fed calves after oral nitrite and nitrate administration

There is marked endogenous production of nitrate in young calves. Here we have studied the contribution of exogenous nitrate and nitrite to plasma concentrations and urinary excretion of nitrite and nitrate in milk-fed calves. In experiment 1, calves were fed 0 or 200 &mgr;mol nitrate or nitrite/kg(0.75) or 100 &mgr;mol nitrite plus 100 &mgr;mol nitrate/kg(0.75) with milk for 3 d. In experiment 2, calves were fed 400 &mgr;mol nitrate or nitrite/kg(0.75) with milk for 1 d. Plasma nitrate rapidly and comparably increased after feeding nitrite, nitrate or nitrite plus nitrate. The rise of plasma nitrate was greater if 400 than 200 &mgr;mol nitrate or nitrite/kg(0.75) were fed. Plasma nitrate decreased slowly after the 3-d administration of 200 &mgr;mol nitrate or nitrite/kg(0.75) and reached pre-experimental concentrations 4 d later. Urinary nitrate excretions nearly identically increased if nitrate, nitrite or nitrite plus nitrate were administered and excreted amounts were greater if 400 than 200 &mgr;mol nitrate or nitrite/kg(0.75) were fed. After nitrite ingestion plasma nitrite only transiently increased after 2 and 4 h and urinary excretion rates remained unchanged. Plasma nitrate concentration remained unchanged if milk was not supplemented with nitrite or nitrate. Nitrate concentrations were stable for 24 h after addition of nitrite to full blood in vitro, whereas nitrite concentrations decreased within 2 h. In conclusion, plasma nitrate concentrations and urinary nitrate excretions are enhanced dose-dependently by feeding low amounts of nitrate and nitrite, whereas after ingested nitrite only a transient and small rise of plasma nitrite is observed because of rapid conversion to nitrate.     

Relationship of nitrate supply with growth rate, plasma membrane-bound and cytosolic nitrate reductase, and tissue nitrate content in tobacco plants

cNR, cytosolic nitrate reductase
PM-NR, plasma membrane-bound nitrate reductase

Activities of plasma membrane-bound nitrate reductase (PM-NR) and cytosolic nitrate reductase (cNR) in tobacco (Nicotiana tabacum L. cv. Samsun) are regulated differently, depending upon the nitrate supply to the culture medium (in sand culture). The cNR activity of roots was higher at low nitrate concentrations with the maximum at 5 mM nitrate supply and declined to low values beyond 5 mM . In contrast, the PM-NR activity of roots increased with higher nitrate concentrations with the maximum at 25 mM nitrate and clearly decreased only at 40 mM . This high PM-NR activity correlated with a low growth rate and might be one of the responses to excess nitrate. Internal nitrate and total nitrogen content of the tissues, however, showed a relative minimum in shoots and in roots of between 15 and 25 mM external nitrate. With declining PM-NR activities beyond 25 mM external nitrate, the nitrate content in the tissue increased indicating an inverse relationship between tissue nitrate content and root PM-NR activity. In leaves both NR activities (cNR and PM-NR) correlated with the internal nitrate content, but with a different response at low nitrate.     

Development of accelerated net nitrate uptake : effects of nitrate concentration and exposure time

Upon initial nitrate exposure, net nitrate uptake rates in roots of a wide variety of plants accelerate within 6 to 8 hours to substantially greater rates. Effects of solution nitrate concentrations and short pulses of nitrate (≤1 hour) upon `nitrate-induced' acceleration of nitrate uptake in maize (Zea mays L.) were determined. Root cultures of dark-grown seedlings, grown without nitrate, were exposed to 250 micromolar nitrate for 0.25 to 1 hour or to various solution nitrate concentrations (10-250 micromolar) for 1 hour before returning them to a nitrate-free solution. Net nitrate uptake rates were assayed at various periods following nitrate exposure and compared to rates of roots grown either in the absence of nitrate (CaSO₄-grown) or with continuous nitrate for at least 20 hours. Three hours after initial nitrate exposure, nitrate pulse treatments increased nitrate uptake rates three- to four-fold compared to the rates of CaSO₄-grown roots. When cycloheximide (5 micrograms per milliliter) was included during a 1-hour pulse with 250 micromolar nitrate, development of the accelerated nitrate uptake state was delayed. Otherwise, nitrate uptake rates reached maximum values within 6 hours before declining. Maximum rates, however, were significantly less than those of roots exposed continuously for 20, 32, or 44 hours. Pulsing for only 0.25 hour with 250 micromolar nitrate and for 1 hour with 10 micromolar caused acceleration of nitrate uptake, but the rates attained were either less than or not sustained for a duration comparable to those of roots pulsed for 1 hour with 250 micromolar nitrate. These results indicate that substantial development of the nitrate-induced accelerated nitrate uptake state can be achieved by small endogenous accumulations of nitrate, which appear to moderate the activity or level of root nitrate uptake.     

Nitrate Reductase Activity in Maize (Zea mays L.) Leaves: I. Regulation by Nitrate Flux

The roles that leaf nitrate content and nitrate flux play in regulating the levels of nitrate reductase activity (NRA) were investigated in 8- to 14-day old maize (Zea mays L.) plants containing high nitrate levels while other environmental and endogenous factors were constant. The nitrate flux of intact plants was measured from the product of the transpiration rate and the concentration of nitrate in the xylem. NRA decreased when the seedlings were deprived of nitrate. The nitrate flux and the leaf nitrate content also decreased. When nitrate was resupplied to the roots, all three parameters increased.     

Nitrate uptake and reduction in higher and lower plants

The nitrogen compounds nitrate and ammonium are the minerals that plants need in large quantities and which limit their growth in temperate zones. The nitrate assimilation pathway starts with nitrate uptake followed by nitrate reduction resulting in ammonium which is fixed into the amino acids glutamine and glutamate in most plants. This review concentrates on nitrate uptake and nitrate reduction with respect to higher and lower plants. The physiology and the progress in molecular approaches of both processes are considered. For nitrate uptake the well‐established uptake systems are discussed and special attention is drawn to nitrate sensing and the nitrate carrier. Knowledge, particularly on nitrate sensing is rare, but it seems to be the first step in a signal transduction chain triggered by nitrate. Therefore further work should consider this topic more frequently. For nitrate reductase the focus is on the post‐translational modification as a regulatory tool for nitrate assimilation, on the intersections of carbon and nitrogen metabolism and on the molecular approaches. A few remarks on how environmental conditions affect nitrate assimilation are also included. Further progress is needed to understand the transduction of positive and negative signals from the environment affecting the expression of genes coding for the nitrate assimilating pathway.     

Nitrogen and Nitrate Accumulation in Species Having Different Relationships Between Nitrate Uptake and Reduction

Species differ in the relationship of nitrate reductase activityto nitrate uptake. In Capsicum annuum different diurnal patternsof leaf nitrate reductase activity and nitrate uptake have beenreported. As a consequence, the relationship of free nitratein the plant to nitrate supplied has a higher level of significancethan has reduced nitrogen to nitrate supplied. In Zea mays ithas been reported that leaf nitrate reductase activity respondsdirectly to nitrate translocation to the leaf and in this speciesthe relationship of greatest significance is reduced nitrogencontent to nitrate supplied. In both species, and also in Cucumis melo, the proportion oftotal plant free nitrate and reduced nitrogen in the roots decreases,and in the stem increases, with increasing nitrate supplied. The accumulation of free nitrate in leaves is accompanied bya quantitatively different relationship between reduced nitrogenand dry weight compared to leaves not accumulating nitrate. Capsicum annuum. L., Cucumis melo L., melon, Zea mays L., maize, sweet corn, nitrate reductase, nitrate uptake     

Purification and further characterization of the second nitrate reductase of Escherichia coli K12

Two nitrate reductases, nitrate reductase A and nitrate reductase Z, exist in Escherichia coli. The nitrate reductase Z enzyme has been purified from the membrane fraction of a strain which is deleted for the operon encoding the nitrate reductase A enzyme and which harbours a multicopy plasmid carrying the nitrate reductase Z structural genes; it was purified 219 times with a yield of about 11%. It is an Mr-230,000 complex containing 13 atoms iron and 12 atoms labile sulfur/molecule. The presence of a molybdopterin cofactor in the nitrate reductase Z complex was demonstrated by reconstitution experiments of the molybdenum-cofactor-deficient NADPH-dependent nitrate reductase activity from a Neurospora crassa nit-1 mutant and by fluorescence emission and excitation spectra of stable derivatives of molybdoterin extracted from the purified enzyme. Both nitrate reductases share common properties such as relative molecular mass, subunit composition and electron donors and acceptors. Nevertheless, they diverge by two properties: their electrophoretic migrations are very different (RF of 0.38 for nitrate reductase Z versus 0.23 for nitrate reductase A), as are their susceptibilities to trypsin. An immunological study performed with a serum raised against nitrate reductase Z confirmed the existence of common epitopes in both complexes but unambiguously demonstrated the presence of specific determinants in nitrate reductase Z. Furthermore, it revealed a peculiar aspect of the regulation of both nitrate reductases: the nitrate reductase A enzyme is repressed by oxygen, strongly inducible by nitrate and positively controlled by the fnr gene product; on the contrary, the nitrate reductase Z enzyme is produced aerobically, barely induced by nitrate and repressed by the fnr gene product in anaerobiosis.     

Influence of fruit load and environmental factors on nitrate reductase activity and on concentration of nitrate and carbohydrates in leaves of eggplant (Solanum melongena)

Both the in vivo (+ nitrate) nitrate reductase (NR) activity (leaf disks incubated in the presence of KNO₃) and the in vivo (? nitrate) NR activity (leaf disks incubated without KNO₃) in leaves of eggplant (Solanum melongena L. cv. Bonica) were affected by rapidly growing fruits. Plants with a fruit load showed more pronounced diurnal variation in (+ nitrate) NR activity and higher (? nitrate) NR activity than plants without fruit. The higher (? nitrate) NR activity was accompanied by higher nitrate and lower sucrose and starch contents of leaves. The more pronounced diurnal changes in (+ nitrate) NR activity were paralleled by more pronounced diurnal variation in carbohydrate content of leaves. Fruit removal led to a decrease in both (? nitrate) NR activity and nitrate concentration in leaves, while the carbohydrate content increased. Plants supplied with ammonium instead of nitrate showed only slightly lower (+ nitrate) but no (? nitrate) NR activity. As for plants treated with nitrate, diurnal changes in (+ nitrate) NR activity were most pronounced in leaves of plants with fruit and this again was paralleled by a more pronounced diurnal variation in the carbohydrate concentration in the leaves. Increasing the oxygen level of the atmosphere to 50% led to a dramatic decrease in the (+ nitrate) NR activity and to an increase in both (? nitrate) NR activity and nitrate concentration, which was accompanied by decreasing carbohydrate contents of the leaves. Low light intensities and extended dark periods caused similar changes in NR activity and nitrate and carbohydrate concentrations in leaves. Increasing the nitrate concentration in the nutrient solution led to a rise in (+ nitrate) and (? nitrate) NR activity, but only the (? nitrate) NR activity paralleled the nitrate concentration in the leaves. This increase in the nitrate concentration was accompanied by a decrease in the carbohydrate content of the leaves. It is concluded that the level of and the diurnal changes in both (+ nitrate) and (? nitrate) NR activity and the concentration of nitrate in the leaves are dependent upon their carbohydrate status.     

The partitioning of nitrate assimilation between root and shoot of higher plants

Abstract The partitioning of nitrate assimilation between root and shoot of higher plant species is indicated by the relative proportions of total plant nitrate reductase activity (NRA) in the two plant parts and the relative concentrations of nitrate and reduced N in the xylem sap. These have been collated here from the literature and temperate and tropical species compared. Both the distribution of NRA and xylem sap nitrate: reduced N indicate that the following four generalizations can be made.

• 1 Temperate, perennial species growing in low external nitrate concentrations (about 1 mol m^?3) carry out most of their nitrate assimilation in the root. As external nitrate concentration increases (in the range found in agricultural soils, 1–20 mol m^?3), shoot nitrate assimilation becomes increasingly important.
• 2 Temperate, annual legume species growing in low external nitrate concentrations carry out most of their nitrate assimilation in the root. Shoot nitrate assimilation increases in importance as external nitrate concentration is increased.
• 3 Temperate, annual non-legume species vary greatly in their partitioning of nitrate assimilation between root and shoot when growing in low external nitrate concentrations. Regardless of the proportion carried out in the root at low external nitrate concentrations, nitrate assimilation in the shoot becomes increasingly important as external nitrate concentration is increased.
• 4 Tropical and subtropical species, annual and perennial, carry out a substantial proportion of their nitrate assimilation in the shoot when growing in low external nitrate concentrations. The partitioning of nitrate assimilation between root and shoot remains constant as external nitrate concentration increases.

It is proposed that a greater proportion of nitrate assimilation occurs in the shoot when an increase in the rate of nitrate uptake does not induce an increase in NR level in the root. Thus, a greater proportion of the nitrate taken up remains unassimilated and is passed into the xylem. A constant partitioning of nitrate assimilation between root and shoot is achieved by balancing NR levels in the root with rates of nitrate uptake. The advantages and disadvantages of assimilating nitrate in either the root or shoot are discussed in relation to temperate and tropical habitats.     

Metabolite control of squash nitrate reductase

Nitrate reductase (EC 1.6.6.1) catalyzes the pyridine nucleotide-linked reduction of nitrate to nitrite in higher plants. We have shown that in squash (Cucurbita maxima Duchesne var. Buttercup), exogenous nitrate increases nitrate reductase activity by increasing steady-state levels of nitrate reductase protein, while glutamine diminishes nitrate reductase activity both by decreasing steady-state levels of nitrate reductase protein and by decreasing cellular nitrate concentrations in plant cells. Other amino acids affect nitrate reductase similarly to glutamine; other metabolites tested including nitrate did not cause major perturbations in the synthesis of other cellular proteins. Thus, it appears that the effects of nitrate and reduced nitrogen compounds on enzymes of the nitrate assimilatory pathway are highly specific for these enzymes, and have little effect on other cellular proteins.     

Nitrate uptake and induction of nitrate reductase in excised corn roots

The characteristics of nitrate uptake and induction of nitrate reductase were studied in excised roots of corn (Zea mays L.). Upon initial exposure to nitrate, the low initial rate of nitrate uptake gradually increased until a steady uptake rate was achieved in 1 to 2 hours depending on the NO(3) (-) concentration. The pattern was observed over a wide range (0.2-5 mm) of nitrate concentrations and was independent of the accompanying cation.The nitrate uptake pattern as a function of increasing external nitrate concentrations (0.2-50 mm) followed saturation type kinetics. The reciprocal plot of the data was not linear but hyperbolic, indicating that more than one Km for nitrate uptake can be resolved from the data. This suggests the existence of either one carrier system with changing kinetic constants or the existence of dual uptake systems. The pattern of induction of nitrate reductase was coincident with the pattern of nitrate uptake as a function of time and increasing nitrate concentrations. The rate of induction of nitrate reductase was regulated by the rate of nitrate flux.Washing the roots for 2 hours enhances nitrate uptake by 2.5-fold over the nonwashed tissue. The presence of nitrate in the washing solution leads to further (3.5-fold over control) increases in the rate of nitrate uptake supporting the contention that nitrate plays a specific role in the induction of the inducible nitrate carrier independent of the washing effect.     

Nitrate reductase activity is required for nitrate uptake into fungal but not plant cells

The ability to transport net nitrate was conferred upon transformant cells of the non-nitrate-assimilating yeast Pichia pastoris after the introduction of two genes, one encoding nitrate reductase and the other nitrate transport. It was observed that cells of this lower eukaryote transformed with the nitrate transporter gene alone failed to display net nitrate transport despite having the ability to produce the protein. In addition, loss-of-function nitrate reductase mutants isolated from several nitrate-assimilating fungi appeared to be unable to accumulate nitrate. Uptake assays using the tracer (13)NO(3)(-) showed that nitrate influx is negligible in cells of a nitrate reductase null mutant. In parallel studies using a higher eukaryotic plant, Arabidopsis thaliana, loss-of-function nitrate reductase strains homozygous for both NIA1 insertion and NIA2 deletion were found to have no detectable nitrate reductase mRNA or nitrate reductase activity but retained the ability to transport nitrate. The reasons for these fundamental differences in nitrate transport into the cells of representative members of these two eukaryotic kingdoms are discussed.     

Factors influencing nitrate depletion in a rural stream

A mass balance procedure was used to analyze rates of nitrate depletion in three adjacent reaches of West Duffin Creek, Ontario, Canada. Daily nitrate losses in individual reaches were highly variable (0.5–24 kg N) during low and moderate stream flows in May–October, 1982–1985. Nitrate removal efficiency (nitrate loss as a % of nitrate input) showed a rapid exponential decline with increased nitrate inputs to each reach. Nitrate losses and nitrate removal efficiency also had a significant negative correlation with stream discharge. The association of large nitrate loads with high stream discharge reduced the nitrate removal capacity of the stream because of shorter residence times and a higher ratio of water volume to stream bed area. Water temperature exhibited a significant positive correlation with nitrate loss which may reflect increased denitrification at higher temperatures.Variations in nitrate losses and nitrate removal efficiency between the three reaches were highly influenced by differences in water residence time. Standarized nitrate losses with respect to water residence time revealed a longitudinal decline in nitrate depletion between the reaches which was associated with a downstream decrease in stream nitrate concentration and in the organic carbon content of fine textured sediments from pool habitats.     

小麦叶内硝酸还原的代谢库

随营养液中No_3~-浓度升高,叶片内No_3~-总量、代谢库大小(NIPS)及硝酸还原酶(NR)活性均升高,其中MPS与NK活性呈同步变化;No_3~-浓度达2.0mmol/L时,两者趋于稳值;若再增加NO_3~-浓度,则被吸收的NO_3~-积累于液泡中,而代谢库中NO_3~-含量(MPS)与NO_3~-总量之比有一定程度降低。低氮(NO_3~-浓度为1.0 mmol/L)情况下,反应液中无NO_3~-时,叶片内NR活性品种间有差异,但在50 mmol/L NO_3~-反应液中则品种间无差异;NK活性高的品种鲁麦8号及品种321叶内有大的NO_3~-代谢库,反应液中NO_3~-对NR活性刺激程度低,代谢库NO_3~-含量与叶NO_3~-总量之比高,而叶组织长时间反应过程中其NR活性衰减速率低。