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.     

Nitrite Uptake Patterns in Wheat Seedlings as Influenced by Nitrate and Ammonium

Nitrite and nitrate uptake by wheat (Triticum vulgare) from 0.5 mM potassium solutions both showed an apparent induction pattern characterized by a slow initial rate followed by an accelerated rate. The accelerated phase was more rapid for nitrate uptake, was initiated earlier, and was seriously restricted by the presence of equimolar nitrite. The accelerated phase of nitrite uptake was restricted by nitrate to a lesser extent. The two anions seem not to be absorbed by identical mechanisms. Ammonium pretreatments or prior growth with ammonium had relatively little influence on the pattern of nitrite uptake. However, prior growth with nitrate eliminated the slow initial phase and induced development of the accelerated phase of nitrite uptake. A beneficial effect was noted after 3 h nitrate pretreatment and full development had occurred by 12 h nitrate pretreatment. The evidence suggests that a small amount of tissue nitrite, which could be supplied either by absorption or by nitrate reduction, was specifically required for induction of the accelerated phase of nitrite uptake. Cycloheximide (2 μg ml^?1) seriously restricted development of the accelerated phase of nitrite uptake, but its effect was not as severe when it was added after the accelerated phase had been induced by prior exposure to nitrite or nitrate. However, translocation of ¹⁵N from the absorbed nitrite was sharply decreased under the latter conditions, indicating a difference in sensitivity of the uptake and translocation processes to cycloheximide. Potassium uptake was greater from KNO₃ than from KNO₂ and in both instances it was enhanced during the early stages of the accelerated phase of anion uptake. Moreover, addition of NaNO₃ to KNO₂ substantially increased potassium uptake. A coupling between anion and potassium uptake was therefore evident, but the coupling was not obligatory because the accelerated phase of nitrite uptake could occur in absence of rapid potassium uptake.     

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.     

Accelerated Nitrate Uptake in Wheat Seedlings: Effects of Ammonium and Nitrite Pretreatments and of 6-Methylpurine and Puromycin

The experiments reported herein had two objectives. One was to determine if the slow rate of nitrate uptake which occurs upon initial exposure of nitrogen-depleted wheat (Triticum vulgare cv. Knox) plants to nitrate was the result of insufficient reduced nitrogen. The second was to determine the impact of restrictions in ribonucleic acid or protein synthesis on both nitrate uptake and nitrate reduction. Pretreatments of 14-day-old seedlings for a few hours in ammonium or nitrite did not result in an enhancement of the initial slow rate of nitrate uptake. Growth for two additional weeks in ammonium also failed to eliminate the induction period. The evidence indicates that the presence of nitrate, rather than a product of its reduction, was required to initiate development of the accelerated rate of nitrate uptake. Puromycin (400 μg ml^?1) and 6-methylpurine (0.5 mM) prevented development of the accelerated phase of nitrate uptake. With both compounds, the relative restriction of nitrate uptake was greater than that of nitrate reduction as revealed by incorporation of ¹⁵N from labeled nitrate into reduced forms. The proportion of reduction which occurred in the root system under the imposed treatments could not be delineated precisely, preventing an unequivocal determination of the extent to which the two processes are coupled in the root system. The data nevertheless indicate nitrate reduction was closely associated with nitrate uptake. Accumulation of nitrate in the shoots was markedly restricted in presence of 6 methylpurine. This effect most likely was a result of a severe restriction in the translocation of nitrate into the xylem, rather than an increase in the reduction rate in the shoots.     

Physiological and biochemical characterization of chlorate-resistant mutants ofAnabaena doliolum

Chlorate-resistant mutants of the filamentous cyanobacterium,Anabaena doliolum, were isolated by N-methyl-N-nitro-N-nitrosoguanidine (MNNG)¹ mutagenesis. Three classes of mutants were obtained that were altered either in the nitrate uptake activity or nitrate reductase enzyme activity or both. These results suggest that the genetic determinant of the uptake system was distinct from that of the reductase system.Uptake studies of nitrite and ammonium and rate of nitrite reductase activity in the mutants revealed that the nitrite and ammonium metabolisms were not affected by this mutation.Both nitrate and chlorate acted like a pair of antagonists, with nitrate protecting the growth against chlorate with increase in its concentration; similarly, increasing chlorate concentrations counteracted the growth-protective action of nitrate.     

Nitrate Uptake during Recovery from Nitrogen Deficiency

Two-week-old nitrogen-deficient wheat plants attained a high rate of nitrate uptake on the first day of exposure to nutrient solutions supplemented with KNO₃. Ammonium uptake from similar solutions supplemented with NH₄NO₃ was also high during the first day of exposure, but nitrate uptake from this solution was lower than from the KNO₃ treatment. During the next two to three days there was a progressive decrease in uptake of both nitrogen ions. A steady increase in uptake then occurred as the plants fully recovered from the nitrogen-deficient state. The transient low nitrate uptake after three or four days of exposure to KNO₃ was not due to an excessive accumulation of nitrate in the tissue, nor to a failure in nitrate reduction as indicated by the rate of nitrate accumulation relative to the uptake rate. Nitrogen supplied as ¹⁵N-nitrite during the low uptake period was effectively incorporated into organic forms and effectively translocated to the shoots. Failure of the root tissue to increase in soluble carbohydrates during illumination was characteristic of the low uptake period. This contrasted with an increase in root soluble carbohydrates in the light during rapid uptake associated with full recovery from the nitrogen-deficient state. It is concluded that carbohydrate translocation to the root system was insufficient during the intermediate recovery period for optimal nitrate uptake, although it was sufficient for effective reduction and translocation of nitrate and reduced nitrogen. Ammonium uptake from NH₄NO₃ was restricted during darkness by the third day whereas there was little difference between light and dark periods in nitrate uptake from KNO₃ until about the sixth day of recovery. The extent to which ammonium restricted nitrate uptake increased progressively for two or three days following which a lessening influence seemed evident, and the effects were not directly associated with the rate of ammonium uptake.     

The Kinetics of Chlorate Uptake by XD Tobacco Cells

The uptake of [³⁶Cl]chlorate by the 14U variant of the XD cell line of Nicotiana tobaccum L. cv Xanthi was investigated to examine the use of chlorate as a nitrate analog in transport studies. The kinetics of chlorate uptake against concentration was complex. Evidence was obtained, e.g., by means of nitrate competition, that these kinetics could be resolved into two components indicating the existence of two influx mechanisms: a saturable high affinity transport system (HATS) and a low affinity transport system (LATS) that showed first order kinetics. HATS has an apparent K_m for chlorate of 0.3 millimolar, and a marked pH dependence. The V_max dropped about fivefold as the pH was changed from the optimum pH (5.5-6.5), while the K_m remained virtually unchanged. The activity of HATS was completely inhibited by 15 millimolar nitrate and was less sensitive to chloride. LATS was inhibited by chloride and showed some inhibition by nitrate. It was concluded that [³⁶Cl]chlorate can be used as an analog for nitrate uptake studies only in a limited low concentration range where HATS is the main route for chlorate influx.     

Nitrate reductase deficient cell lines from diploid cell cultures and lethal mutant M2 plants of Arabidopsis thaliana

Summary Cell suspensions of diploid Arabidopsis thaliana were screened for resistance to chlorate on a medium with ammonium nitrate as the nitrogen source, and after plating on filters to increase the plating efficiency. Thirty-nine lines were selected, four of which were still resistant after two years of subculturing on non-selective medium. Of the latter lines three were nitrate reductase deficient but exhibited some residual nitrate reductase activity; the fourth line showed a high level of enzyme activity. Screening M2-seeds for callus production on selective medium with amino acids as the nitrogen source and chlorate revealed resistant calli in 17 out of 483 M2-groups. Nine well-growing lines, all but one (G3) exhibiting no detectable in vivo nitrate reductase activity, were classified as defective in the cofactor. Two lines (G1 and G3) could be analysed genetically at the plant level. Chlorate resistance was monogenic and recessive. Sucrose gradient fractionation of callus extracts of G1 revealed that a complete enzyme molecule can be assembled. Nitrate reductase activity in G1 could partly be restored by excess molybdenum. It is suggested that G1 is disturbed in the catalytic properties of the cofactor. It appeared that G1 is neither allelic with another molybdenum repairable mutant (B73) nor with another cofactor mutant (B25). Wilting of intact G1 plants could be ascribed to non-closing stomata.     

Chlorate Toxicity and Nitrate Reductase Activity in Tomato Plants

Chlorate damage was studied in tomato plants ( Lycopersicum esculentum cv. Moneymaker) that were supplied with a nitrogen-free nutrient solution or with a nutrient solution, containing either nitrate or ammonium as a nitrogen source. Damage was low in ammonium-fed plants and high in nitrate-fed plants and in nitrogen-less plants. Nitrate reductase activity could be detected in all treatments, although the activity was highest in the nitrate-fed plants.
The hypothesis that chlorate can be used as a substrate by the enzyme nitrate reductase in higher plants, was studied and proved to be true for the tomato plants, as was found earlier for Escherichia and Chlorella . The affinity of the enzyme for chlorate was lower than for nitrate, the K _m being 4 m M and 0.15 m M respectively. Induction of the enzyme by chlorate could not be detected. The enzyme activity was lowered in leaf discs after a 7 h treatment with chlorate and the inhibition was proportional to the chlorate concentration of the medium.
The results were discussed in terms of competition between nitrate and chlorate at the uptake and the enzyme site and with regard to a possible influence of chlorate on synthesis and breakdown of the enzyme.     

Isolation and characterization of nitrate reductase-deficient mutants of Arabidopsis thaliana

Summary Chlorate resistant mutants of Arabidopsis thaliana were isolated, of which 10 exhibited a lowered nitrate reductase activity and 51 were chlorate-resistant because of an impaired uptake of chlorate. The 51 mutants of this type are all affected in the same gene. The mutants with a lowered nitrate reductase activity fall into 7 different complementation groups. Three of these mutants grow poorly on media with nitrate as the sole nitrogen source, while the others apparently can reduce sufficient nitrate to bring about growth. In all cases a low nitrate reductase activity coincides with an enhanced nitrite reductase activity. After sucrose gradient centrifugation of wildtype extracts nitrate reductase is found at the 8S position, whereas cytochrome-c reductase is found both at 4 and 8S positions. It is suggested that the functional nitrate reductase is a complex consisting of 4S subunits showing cytochrome-c reductase activity and a Mo-bearing cofactor. All mutants except B25 are capable of assembling the 4S subunits into complexes which for most mutants have a lower S value and exhibit a lower nitrate reductase activity than the wildtype complexes. Since the mutants B25 and B73 exhibit a low xanthine dehydrogenase activity, the Mo-bearing cofactor is probably less available in these mutants than in the wildtype. B73 appears to be the only mutant which is partly repaired by excessive Mo. The possible role of several genes is discussed.     

Time-course of Nitrate Uptake and Nitrate Reductase Activity in Nitrogen-depleted Dwarf Bean

The rate of nitrate uptake by N-depleted French dwarf bean (Phaseolus vulgaris L. cv. Witte Krombek) increased steadily during the first 6 h after addition of NO₃ -After this initial phase the rale remained constant for many hours. Detached root systems showed the same time-course of uptake as roots of intact plants. In vivo nitrate reductase activity (NRA) was assayed with or without exogenous NO₃^- in the incubation medium and the result ing activities were denoted potential and actual level, respectively. In roots the difference between actual and potential NRA disappeared within 15 min after addition of nitrate, and NRA increased for about 15 h. Both potential and actual NRA were initially very low. In leaves, however, potential NRA was initially very high and was not affected by ambient nitrate (0.1–5 mol m^-3) for about 10 h. Actual and potential leaf NRA became equal after the same period of time. In the course of nitrate nutrition, the two nitrate reductase activities in leaves were differentially inhibited by cycloheximide (3.6 mmol m^-3) and tungstate (1 mol m^-3). We suggest that initial potential NRA reflects the activity of pre-existing enzyme, whereas actual NRA depends on enzyme assembly during NO₃^- supply. Apparent induction of nitrate uptake and most (85%) of the actual in vivo NRA occurred in the root system during the first 6 h of nitrate utilization by dwarf bean.     

Nitrate reduction in the wildtype and a nitrate reductase deficient mutant of Arabidopsis thaliana

A chlorate-resistant mutant B25 of Arabidopsis thaliana (L.) Heinh. was isolated, which has very little or no in vitro nitrate reductase activity and grows poorly on a substrate with nitrate as the sole nitrogen source. The mutation of B25 ( rgn ) is monogenic and recessive, tightly linked to the marker gene an on chromosome 1. Nitrate induces cytochrome- c reductase activity in the mutant but to a lower level than in the wildtype. After sucrose gradient centrifugation the greatest part of the cytochrome- c reductase from induced wildtype is found as 8s type whereas cytochrome- c reductase from B25 under the same conditions is found as 4s type. Nitrate reductase is found at the 8s position. It is suggested that B25 has lost the ability to assemble two 4s subunits showing cytochrome- c reductase activity and a Mo-bearing co-factor into the functional nitrate reductase. Nitrate rather than nitrite is the inducing agent for nitrite reductase, since in B25 nitrite reductase is even more rapidly induced than in the wildtype after addition of nitrate. Both the wildtype and B25 contain a nitrate reductase inhibiting factor when grown on ammonium. This inhibiting factor is a small protein, possibly similar to the nitrate reductase inactivating enzyme reported for other plants.     

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     

Removal of Nitrate from Groundwater by Cyanobacteria: Quantitative Assessment of Factors Influencing Nitrate Uptake

The feasibility of biologically removing nitrate from groundwater was tested by using cyanobacterial cultures in batch mode under laboratory conditions. Results demonstrated that nitrate-contaminated groundwater, when supplemented with phosphate and some trace elements, can be used as growth medium supporting vigorous growth of several strains of cyanobacteria. As cyanobacteria grew, nitrate was removed from the water. Of three species tested, Synechococcus sp. strain PCC 7942 displayed the highest nitrate uptake rate, but all species showed rapid removal of nitrate from groundwater. The nitrate uptake rate increased proportionally with increasing light intensity up to 100 μmol of photons m⁻² s⁻¹, which parallels photosynthetic activity. The nitrate uptake rate was affected by inoculum size (i.e., cell density), fixed-nitrogen level in the cells in the inoculum, and aeration rate, with vigorously aerated, nitrate-sufficient cells in mid-logarithmic phase having the highest long-term nitrate uptake rate. Average nitrate uptake rates up to 0.05 mM NO₃⁻ h⁻¹ could be achieved at a culture optical density at 730 nm of 0.5 to 1.0 over a 2-day culture period. This result compares favorably with those reported for nitrate removal by other cyanobacteria and algae, and therefore effective nitrate removal from groundwater using this organism could be anticipated on large-scale operations.     

Nitrate Utilization by Nitrate Reductase-deficient Barley Mutants

Two nitrate reductase-deficient barley mutants were studied for growth on nitrate and ammonium sources of nitrogen and for resistance to chlorate. Although nitrate reductase-deficient mutants in some species are chlorate-resistant (unable to reduce chlorate to chlorite), the barley mutants used in these studies when grown on nitrate and treated with chlorate were only slightly more resistant to chlorate than the control. When grown to maturity on vermiculite supplemented with either nitrate or ammonium nutrient solutions, the mutants produced as much dry weight and reduced nitrogen per plant as the control. The in vivo and in vitro nitrate reductase activities in the roots and shoots of the mutants grown on nitrate were consistently less than 10% of the control. To avoid the possibility that the mutants received reduced nitrogen from microbial sources, excised embryos were cultured under sterile conditions. Again the mutants were capable of growth and reduced nitrogen accumulation with nitrate as the sole source of nitrogen. In spite of the low apparent nitrate reductase activity, the nitrate reductase-deficient mutants are capable of substantial nitrate reduction.     

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.     

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     

Inhibition of Assimilatory Nitrate Uptake by Ammonium Ions in Azorhizobium caulinodans IRBG 46

Addition of ammonium sulphate at low concentrations to Azorhizobium caulinodans IRBG 46 cells caused an immediate cessation of nitrate uptake activity, which was restored when the added ammonium ions were exhausted from the medium. Blockage of ammonium assimilation by L-methionine sulfoximine did not prevent the negative effect of ammonium on the assimilatory nitrate uptake, thus indicating that ammonium ions per se and not its assimilatory product(s) are actual regulators of assimilatory nitrate uptake.     

Uptake of chlorate and other ions in seedlings of the nitrate-uptake mutant B1 of Arabidopsis thaliana

The accumulation of labelled ions was measured in seedlings of Arabidopsis thaliana (L.) Heynh. grown in submerged cultures. Genotypes used were wild type and the nitrate-uptake mutant B1, which is altered in the uptake of nitrate and chlorate from concentrations above 1 m M when grown under normal conditions. At low and high concentrations of chlorate, chloride, and K⁺, significantly less radioactivity was accumulated in seedlings of B1 than in seedlings of wild type. The influx of sulphate did not decrease in B1. The results indicate that the effect of the mutation in B1 is restricted to the uptake of monovalent ions.