Promotion of Assimilation of Ammonium Ions by Simultaneous Application of Nitrate and Ammonium Ions in Radish Plants

When radish plants were grown in nutrient solutions that containedammonium ions (NH₄⁺) as the sole source of nitrogen, they grewpoorly and accumulated high levels of NH₄⁺ in their leaves.However, radish plants cultured in 5 mM NH₄⁺ plus 1 mM NO₃^–(a ratio of 5 : 1 in forms of nitrogen; referred to as 5:lmix-N)grew well and accumulated very low levels of NH₄⁺ in their leaves.After radish plants were cultured in solutions that containedNO₃^–, or NH₄⁺, or 5: lmix-N for a week, they were thensupplied with the same nitrogen source labeled with ¹⁵N forone day. The uptake of ¹⁵N from labeled NH₄⁺ into total nitrogenwas the highest in plants supplied with 5:1mix-N. These plantsconverted far fewer labeled NH₄⁺ into free NH₄⁺ than did NH₄⁺-fedplants, but converted many more labeled NH₄⁺ into the insolublefraction than did NH₄⁺- or NO₃^–-fed plants. The presence of a small amount of nitrate was shown to stimulatethe assimilation of ammonium ions and the synthesis of proteins. (Received October 26, 1988; Accepted January 24, 1989)     

The Assimilation of Nitrogen from Ammonium Salts and Nitrate by Fungi

1. The assimilation of inorganic nitrogen by Scopulariopsis brevicaulisand some physiologically similar species has been studied. Theirfailure to assimilate completely from ammonium sulphate hasbeen shown to be due to the fall in pH of the medium inducedby the initial uptake of ammonia.
2. Complete assimilation ofammonia takes place in the presenceof the neutral salts ofeach of thirteen organic acids investigated.The organic acidsact primarily through their buffering effectwhich preventsor slows down the fall in pH. They are not specificallyrequiredfor ammonia assimilation by these fungi and can beeffectivelyreplaced by certain inorganic buffers.
3. The influence of severalexternal factors on the rate of assimilationof ammonia, nitrate,and nitrite has been studied in S. brevicaulis.In correspondingconditions the mycelium assimilates ammoniamore rapidly thannitrate over a wide range of conditions.
4. Ammonia, even invery low concentration, completely suppressesnitrate assimilationwhen both sources of nitrogen are presenttogether. Nitrite,however, is assimilated simultaneously withammonia. It is thereforeconcluded that ammonia blocks the reductionof nitrate to nitriteby the fungus.
5. The suppression of nitrate assimilation inthe presence of ammoniais common to many mould fungi besidesS. brevicaulis, and isbelieved to have adaptive significancein natural habitats.
6. The nitrate-reducing and assimilatingsystem is formed, evenwhen S. brevicaulis is grown in completeabsence of nitrate(ammonia medium with organic acid). It comesinto action rapidlywhen the inhibiting effect of ammonia isremoved. Similarly,nitrate-grown mycelium is capable of assimilatingammonia atmaximal rate without any adaptive lag.

     

Nitrate Uptake and Assimilation by Wheat Seedlings during Initial Exposure to Nitrate

Nitrate uptake, reduction, and translocation were examined in intact, 14-day-old, nitrogen-depleted wheat (Triticum vulgare var. Knox) seedlings during a 9-hour exposure to 0.2 mm Ca (NO(3))(2). The nitrate uptake rate was low during the initial 3-hour period, increased during the 3- to 6-hour period, and then declined. By the 3rd hour, 14% of the absorbed nitrate had been reduced, and this increased to 36% by the 9th hour. Shoots accumulated reduced (15)N more rapidly than roots and the ratio of reduced (15)N to (15)N-nitrate was higher in the shoots. A significant proportion of the total reduction occurred in the root system under these experimental conditions. Accumulation of (15)N in ethanol-insoluble forms was evident in both roots and shoots by the 3rd hour and, after 4.5 hours, increased more rapidly in shoots than in roots.An experiment in which a 3-hour exposure to 0.2 mm Ca ((15)NO(3))(2) was followed by a 12-hour exposure to 0.2 mm Ca ((14)NO(3))(2) revealed a half-time of depletion of root nitrate of about 2.5 hours. A large proportion of this depletion, however, was due to loss of (15)N-nitrate to the ambient (14)N-nitrate solution. The remaining pool of (15)N-nitrate was only slowly available for reduction. Total (15)N translocation to the shoot was relatively efficient during the first 3 hours after transfer to Ca ((14)NO(3))(2) but it essentially ceased after that time in spite of significant pools of (15)N-nitrate and alpha-amino-(15)N remaining in the root tissue.     

Nitrate and Ammonium Assimilation by Roots of Maize (Zea mays L.) Seedlings as Investigated by In Vivo 15N-NMR

¹⁵N-Nuclear Magnetic Resonance (NMR) was used to study nitrogenassimilation in apices of maize roots in vivo, perfused eitherwith ¹⁵NO     

Response of Ammonia Assimilation in Cucumber Seedlings to Nitrate Stress

The influence of increased nitrate concentration—14 (control) and 140 mmol L⁻¹ (T)—in hydroponic culture on ammonia assimilation in cucumber (Cucumis sativus L. cv. Xintaimici) seedlings was investigated. The results showed that NH₃ accumulation in the roots and leaves of T seedlings increased significantly, indicating that NH₃ toxicity might be involved in nitrate stress. Under control conditions, GS and GOGAT activity were much higher in the leaves than in the roots, whereas GDH activity was much higher in the roots than in the leaves. Correlation analysis showed that NH₃ concentration had a strong negative linear relationship with GDH activity in the roots but had a strong negative linear relationship with GS and GOGAT activity in the leaves. These results indicate that NH₃ might be assimilated primarily via GDH reaction in the roots and via GS/GOGAT cycle in the leaves. Short-term nitrate stress resulted in the increase of GS and GOGAT activity in the roots and GDH activity in the leaves of T seedlings, indicating possible shifts in ammonia assimilation from the normal GDH pathway to GS/GOGAT pathway in the roots and from the normal GS/GOGAT pathway to the GDH pathway in the leaves under nitrate stress, but with the increase of treatment time, GS, GOGAT, and GDH activity in the roots and leaves of T seedlings decreased possibly due to low water potential and NH₃ toxicity.     

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.     

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.     

The Effects of Irradiance on Uptake and Assimilation of Nitrate by Young Barley Seedlings

The nitrogen economy of barley plants growing in a range ofirradiances from full shade (less than 0·5 W m^–2)to 119 W ^m–2 has been examined by analysing levels oftotal, organic and nitrate nitrogen, and by determining nitratereductase activity in leaf extracts. It has been confirmed thatroot growth is reduced in low irradiances which are also associatedwith a lower level of total nitrogen in the plant, and hencewith a lower uptake of nitrate. In all parts of the plant thelevel of organic nitrogen is higher in high light intensitybut nitrate-nitrogen as a proportion of the total is greatestin low irradiances. In the first leaf accumulation of free nitrateis substantially greater in low irradiances. The data indicate a higher level of nitrate assimilation inhigh irradiances and nitrate reductase activity in leaf extractsis higher in such conditions. When the first leaf is shadednitrate reductase activity falls to undetectable levels afterabout 4 days, but in the case of the second leaf, where thisis shaded, some reductase activity is always found, althoughthis is substantially less than that in unshaded conditions. It is concluded that in vitro rates of nitrate reduction mayover-estimate nitrate assimilation determined as increase inorganic nitrogen.     

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.     

The Effect of Nitrate and Ammonium Feeding on Carbon Dioxide Assimilation in Maize

The effect of nitrogen nutrition on the pattern of ¹⁴CO₂ fixationduring photosynthesis by young maize plants was studied. Nitratefeeding increased the incorporation of ¹⁴C into malate and certainamino acids (particularly aspartate) and decreased that intosucrose and starch. Ammonium nutrition caused similar, but notidentical, changes, the major difference being a much smallereffect on incorporation into malate. Tungstate applied to theplants prior to nitrate almost completely inhibited the formationof nitrate reductase and eliminated most, but not all, of theeffects of nitrate on the fixation pattern. The results obtainedare discussed in terms of: (i) more reduced nitrogen as substratefor amino acid synthesis; (ii) the changes in pH and ionic balancecaused by the process of nitrate reduction to ammonium; and(iii) specific enzyme activation or inhibition by nitrate orammonium ions.     

Effect of Previous Nitrate Deprivation on 15N-nitrate Absorption and Assimilation by Wheat Seedlings

Nitrate uptake and assimilation were examined in intact 18 days old wheat (Triticum aestivum, cv Capitole) seedlings either permanently grown on nitrate (high-N seedlings) or N-stressed by transfer to an 0 N-solution for the final 7 days (low-N seedlings). The N-stressed seedlings were characterized by a lower organic N content (2.5 mg instead of 4.9 mg per seedling) and an increased root dry weight.The seedlings received ¹⁵NO₃K for 7 h in the light. Nitrate uptake was 2.8 times higher in low-N than in high-N seedlings. The assimilation rate was 35 and 16 μmol NO₃^?·^h?1· g^?1 dry weight respectively. Partitioning of NO₃^? to reduction and assimilation was the very same in both kinds of seedlings. The results support the view that 50 % of the nitrate reduction in Triticum aestivum, cv Capitole could be achieved in the roots.The present observations are interpreted as evidence that factors closely associated with the seedling N-status may have a major role in regulating NO₃^? uptake and assimilation. In low-N seedlings, the high amount of carbohydrates in roots may add its stimulus to the specific inducing effect of nitrate whereas in high-N seedlings, excess of nitrate or amino-acids may set the pace by negative feedback control.     

Outgrowth of Bacillus megaterium Spores in the Presence of Nitrate and Ammonium Ions

Nitrate or ammonium ions, but not nitrite ion, supplied the nitrogen required for outgrowth of Bacillus megaterium QM B1551 spores through the first cell division. This outgrowth was markedly favored by K⁺.     

Stimulation of Nitrate and Nitrite Efflux by Ammonium in Barley (Hordeum vulgare L.) Seedlings

The inhibitory effect of NH4+ on net NO3- uptake has been attributed to an enhancement of efflux and, recently, to an inhibition of influx. To study this controversy, we devised treatments to distinguish the effects of NH4+ on these two processes. Roots of intact barley (Hordeum vulgare L.) seedlings, uninduced or induced with NO3- or NO2-, were used. Net uptake and efflux, respectively, were determined by following the depletion and accumulation in the external solutions. In roots of both uninduced and NO2- -induced seedlings, NO3- efflux was negligible; hence, the initial uptake rates were equivalent to influx. Under these conditions, NH4+ had little effect on NO3- uptake (influx) rates by either the low- or high-Km uptake systems. In contrast, in plants preloaded with NO3-, NH4+ and its analog CH3NH3+ decreased net uptake, presumably by enhancing NO3- efflux. The stimulatory effect of NH4+ on NO3- efflux was a function of external NH4+ and internal NO3- concentration. These results were corroborated by the absence of any effect of NH4+ on NO2- uptake unless the roots were preloaded with NO2-. In this case NH4+ increased efflux and decreased net uptake. Hence, the main effect of NH4+ on net NO3- and NO2- uptake appears to be due to enhancement of efflux and not to inhibition of influx.     

Short-term Studies of the Uptake of Nitrogen by Young Apple Trees after Soil Application of Ammonium Nitrate

Young trees of apple rootstock MM.104 were supplied with ammoniumnitrate (0.4 g in 100 ml water) via the soil at selected timesduring the annual growth cycle. Appreciable amounts of nitrogenwere taken up within 40 h in summer and autumn, leading to increasesin the soluble nitrogen fraction. Corresponding changes werenot detected in the insoluble nitrogen fraction. The increasesin soluble nitrogen were not dependent on carbohydrate status,plant size, root mass or on whether or not shoot extension hadceased. Amounts of increase were profoundly influenced by priornitrogen status as modified by a small pre-treatment boost ofammonium nitrate given in July.     

Contamination of Ammonium-Based Nutrient Solutions by Nitrifying Organisms and the Conversion of Ammonium to Nitrate

Conversion of ammonium to nitrate and contamination by nitrifying organisms are often assumed not to be significant in ammonium-based nutrient solutions. To assess this assumption, maize (Zea mays) and pea (Pisum sativum) were grown under greenhouse conditions in aeroponic, hydroponic, and sand-culture systems containing 2 mM ammonium chloride as the sole nitrogen source and evaluated for the activity of contaminating nitrifying organisms. In all three culture systems, root colonization by nitrifying organisms was detected within 5 d, and nitrate was detected in the nutrient solution within 10 d after seedling transfer. In sand culture, solution nitrate concentration reached 0.35 mM by the end of the 17-d experiment. Consistent with the microbial ammonium oxidation sequence, nitrite was detected earlier than nitrate and remained at lower levels throughout the experiment. Nitrate was found in significant quantities in root and shoot tissues from seedlings grown in ammonium-based nutrient solutions in all of the solution culture systems. Maize seedlings grown in an ammonium-based hydroponic system contained nitrate concentrations at 40% of that found in plants grown in nitrate-based solution. Determination of nitrate (or nitrite) levels in the nutrient solution was the weakest indicator of the activity of nitrifying organisms. A bioassay for the presence of nitrifying organisms in combination with tissue analysis for nitrate was a better indicator of microbial conversion of ammonium to nitrate in nutrient solution culture. The results have implications for the use of ammonium-based nutrient solutions to obtain plants suitable for research on induction of nitrate uptake and reduction or for research using solution culture to compare ammonium versus nitrate fertilization.     

Potential Nitrification as an Indicator of Preferential Uptake of Ammonium or Nitrate by Plants in an Oak Woodland Understorey

The preferences of some woodland understorey species for ammoniumand nitrate were investigated by measuring the potential nitrification(conversion of ammonium to nitrate) in the rhizosphere comparedwith the bulk soil. Less acid-tolerant species, which usuallyprefer nitrate or a mixture of ammonium and nitrate in hydroponicculture, should have a higher potential nitrification in therhizosphere compared to the bulk soil due to a low uptake ofammonium (since ammonium is relatively immobile). Acid-tolerantspecies should have a high uptake of ammonium and thereby loweror equal potential nitrification in the rhizosphere comparedto the bulk soil. The hypothesis was tested in a field investigationof five understorey herb species, Deschampsia flexuosa, Convallariamajalis, Poa nemoralis, Geum urbanum andAegopodium podagrariaperformed in oak forests in southern Sweden. Overall, the twoless acid-tolerant species, Geum urbanum and Aegopodium podagraria,had high potential nitrification in the rhizosphere comparedto the bulk soil (indicating a relatively low uptake of ammonium),whilst the acid tolerant species, Deschampsia flexuosa andConvallariamajalis , had approximately equal potential nitrification inthe rhizosphere compared to the bulk soil (indicating a relativelyhigh uptake of ammonium). In the case of Poa nemoralis, a specieswhich grows in both acid and less acid soils, we found the potentialnitrification in the rhizosphere and in the bulk soil to besimilar at low inorganic nitrogen concentrations, but the difference(rhizosphere > bulk) increased when nitrification in thebulk soil was enhanced (i.e. when the nitrogen availabilityincreased). The potential nitrification in the bulk soil variedbetween 0 and 16 nmol g^-1h^-1and was positively correlated withpH. When species occurred at the same site, the potential nitrificationin the bulk soil tended to be lower for the acid tolerant species.Despite a large variation in potential nitrification, the methodoffers a possibility of measuring the preference of plants forammonium/nitrate in a soil system, under natural conditions.Copyright 2000 Annals of Botany Company Ammonium uptake, nitrate uptake, nitrogen preference, potential nitrification, rhizosphere, Deschampsia flexuosa, Convallaria majalis, Poa nemoralis, Geum urbanum, Aegopodium podagraria     

Some Aspects of Nitrate Assimilation in the Seedlings of Normal and Opaque-2 Mutant of Maize

The effects of 10 mM nitrate on the growth and nitrogenous componentsof Zea mays L. var. W64A wild type (normal) were compared tothose on its opaque-2 (high lysine) mutant during the first10 d of seedling growth at a constant temperature of 26 °Cand with a 16 h photoperiod. Nitrate supply had no effect onthe growth of embryonic axes in both lines till day 6. Growthof both lines was enhanced slightly after that time, however.Increases in 80% (v/v) ethanolsoluble and protein nitrogen werealso observed only after day 4 when the supply of nitrogen fromthe storage proteins in the endosperm was limiting. Nitratehad no effect on the synthesis of chlorophyll during leaf developmentbut it did increase the total chlorophyll in mature and senescingprimary leaves. The increase in nitrogenous components or chlorophyllin opaque-2 was more pronounced than in the normal type. Itmight be related to the lower proline or higher lysine in themutant.     

Inhibition of Nitrate Assimilation in Roots in the Presence of Ammonium: The Moderating Influence of Potassium

Experiments were conducted to investigate the effect of concentrationof NH₄⁺ in nutrient solution on root assimilation of NO₃^–and to determine whether the NH₄⁺NO₃^– interaction wasmodified in the presence of K⁺. Dark-grown, detopped corn seedlings(cv. Pioneer 3369A) were exposed for 8 h to 0.15 mM Ca(NO₃)₂and varying concentrations of (NH₄)₂SO₄ in the absence or presenceof 0.15 mM K₂SO₄. The accelerated phase of NO₃^– uptakeappeared most sensitive to restriction by additions of 0.15mM (NH₄)₂SO₄. In the absence of K⁺, the restriction increasedonly slightly even when solution (NH₄)₂SO₄, was increased from0.15 mM to 12.5 mM which was accompanied by an increase of NH₄⁺in the tissue from about 7.0 to 35 µmol g^–1 fr.wt. of root. Increasing concentrations of solution NH₄⁺ progressivelyinhibited net K+ uptake. At the highest solution NH₄⁺ concentrations,there was an initial net efflux of K⁺ and no net influx occurredduring the treatment period. The severity of the NH₄)SO₄ restrictionof NO₃^– uptake was moderated considerably in the presenceof K⁺ as long as a net influx of K⁺ occurred. However, net influxof K⁺ was not associated with alteration of NH₄⁺ uptake, assimilation,or accumulation in the root tissue. The lack of correlationbetween the severity of restriction of NO₃^– uptake andendogenous NHJ suggested the restriction resulted from an effectexerted by exogenous NH₄⁺ which tended to saturate at lowersolution NHJ concentrations or by inhibitory factors generatedduring assimilation of NH₄⁺. Several mechanisms were postulatedto account for the moderating influence of K⁺. In all experiments,root NO₃^– reduction was restricted by the presence ofambient NH₄⁺. The quantitative decreases in reduction tendedto be less than decreases in NO₃^– uptake and therefore,could result from inhibition solely of uptake with subsequentlimitation in availability of substrate for the reduction process,but the possibility of a direct effect on reduction could notbe excluded.