Effect of a constant supply of different nitrogen sources on protein and carbohydrate content and enzyme activities of Anabaena variabilis cells

The effect of the nitrogen source on carbohydrate and protein contents and on several enzymatic activities involved in the carbon and nitrogen metabolism was studied in Anabaena variabilis ATCC 29413 cells grown under a constant supply of either N, NO⁻₃ or NH⁺₄ at different concentrations. An enhancement of protein content accompanied by a parallel decrease of carbohydrates was observed with increasing NO⁻₃ or NH⁺₄ concentrations in the medium. In cultures containing 0.1 m M NO⁻₃ or 0.1 m M NH⁺₄ nitrogenase (EC 1.18.6.1) activity was 74 and 66%, respectively, of that found in N₂-grown cells. This activity was still present with 1 m M NO⁻₃ or 1 m M NH⁺₄ in the medium and even with 10 m M NO⁻₃, but it was completely inhibited by 5 m M NH⁺₄. Ferredoxin-nitrate reductase (EC 1.7.7.2) activity was detected only in NO⁻₃ grown cells and simultaneously with nitrogenase activity. Increasing concentrations of combined nitrogen in the medium, especially NH⁺₄, promoted a concomitant decline of glutamine synthetase (EC 6.3.1.2), NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42), and NAD⁺-malate dehydrogenase (EC 1.1.1.37) activities, suggesting that these enzymes play an important role in the regulation of carbon-nitrogen metabolism in cyanobacteria.     

Effects of NH+4-N and NO+3-N on growth and metabolism of Sphagnum magellanicum

Photosynthetic CO₂-fixation, chlorophyll content, growth rate and nitrate reductase activity were used to examine the influence of NH⁺₄-N and NO⁻₃-N on Sphagnum magellanicum cultivated under defined conditions in phytotrons. NO⁻₃-concentrations up to 322 μ M were found to be favourable. Increased NH⁺₄ concentrations, however, resulted in growth inhibition and decreased chlorophyll content at concentrations ≧ 255 μ M ; e.g. 600 μ M NH⁺₄ caused a 20% reduction of nitrate reductase activity and net photosynthesis. For raised bog Sphagna an improved standard nutrient solution is proposed with the following ion concentrations (μ M ): 55 Na⁺; 17 K⁺; 95 NH⁺₄; 22 Ca²⁺; 22 Mg²⁺; 2 Fe³⁺; 20 Cl⁻; 100 NO⁻₃; 57 SO^2-₄; 7.4 H₂PO⁻₄; trace elements: A-Z solution (Hoagland) 50 μl 1000 ml⁻¹; pH 5.8.     

Uptake regions of inorganic nitrogen in roots of carob seedlings

Three-week-old seedlings of carob ( Ceratonia siliqua L. cv. Mulata) were grown for 9 weeks under different root temperatures (20, 30 and 40°C) at pH values of 5, 7 and 9 with nitrate or ammonium as nitrogen source. Nitrogen uptake rates were determined by depletion from the medium and decreased with distance from the apex. The decline of nitrogen uptake rates along the roots depended on the form of inorganic nitrogen in the medium as well as on pH and temperature, such that the NO⁻₃ and NH⁺₄ ions were taken up essentially by the root tips (0–2 cm) through processes requiring energy. The uncharged NH₃ species entered passively, through the mature parts of the root (2–10 cm). Root zone temperature and pH affect the NH⁺₄/NH³ equilibrium in the nutrient solution and, consequently, the uptake areas of the root for these ions. Furthermore. while root tip uptake of nitrogen is energy dependent, uptake through mature root areas is essentially passive and seems to depend on a well developed apparent free space.     

The use of compound continuous flow diffusion chemostats to study the interaction between nitrifying and nitrate-reducing bacteria

Abstract The interactions occuring between populations of a nitrate-respiring Vibrio sp. and autotrophic nitrifying bacteria belonging to the genera Nitrosomonas and Nitrobacter have been investigated in a compound bi-directional flow diffusion chemostat at a dilution rate of 0.025 h⁻¹ and a temperature of 25°C. When grown under NO⁻₃ limitation, the Vibrio sp. produced NH⁺₄ as the principal end-product of nitrate respiration, and there was a corresponding significant increase in cell numbers of the Nitrosomonas sp. population, which derived energy by the oxidation of NH⁺₄ to NO⁻₂. Nitrite in turn was used by the Nitrobacter sp. population as an energy source with the concomitant regeneration of NO⁻₃. Under NO⁻₃ excess growth conditions the Vibrio sp. produced NO⁻₂ rather than NH⁺₄ as the major product of NO⁻₃ dissimilation, and growth of the Nitrobacter population was stimulated as increased quantities of NO⁻₂ became available. In contrast, the Nitrosomonas sp. population declined sharply as the energy source NH⁺₄ became limiting. These data demonstrate that defined mixed populations of obligately aerobic nitrifying bacteria and facultatively anaerobic nitrate respiring bacteria can co-exist for extended time periods and operate an internal nitrogen cycle which is energetically beneficial to both populations.     

Changes in nitrogen source modify distribution of excitation energy in the cyanobacterium Phormidium laminosum

In an attempt to clarify the interactions between the available nitrogen source and the photosystems in cyanobacteria, O₂ exchange and fluorescence emission were monitored in spheroplasts and intact cells of the non N₂-fixing cyanobacterium Phormidium laminosum (strain OH-1-p.Cl₁) growing on different nitrogen sources or in the absence of nitrogen. Short-term (time scale of seconds to minutes), NH⁺₄ addition to NO⁻₃-growing or N-starved cells and, to a minor extent, NO⁻₃addition to N-starved cells, induced state 2 transitions both in light and dark. Long term (time scale of days), the fluorescence yield of PSII relative to that of PSII at 77 K was higher in NO⁻₃- than in NH⁺₄ growing cells, and even higher in N-starved cells. In the dark, the plastoquinone pool was more reduced in NH⁺₄- than in NO⁻₃-growing cells. Both PSII and PSI activities and the degree of linking between both photosystems were affected in the long term, so that non-cyclic electron transport decreased in parallel to the ferredoxin requirement to assimilate each nitrogen source. Results indicate that nitrogen metabolism exerts short- and long-term control over the photosynthetic apparatus, which acclimates to the energy requirement of the available nitrogen source.     

Modification of NO−3 metabolism in heterocysts of the N2-fixing cyanobacterium Anabaena 7120 (ATCC27893)

Abstract The utilization of NO⁻₃, NO⁻₂ and NH⁺₄ was studied in whole filaments and isolated heterocysts of Anabaena 7120 (ATCC27893). NO⁻₃- and NO⁻₂-uptake were detectable in whole filaments but not in heterocysts, whereas NH⁺₄-uptake was detectable in both. Activity of NO⁻₃-reductase was present in cell-free extracts of whole filaments but not of heterocysts, whereas activities of NO⁻₂-reductase and glutamine synthetase were present in both. NO⁻₃-uptake and reductase activities could not be induced in heterocysts even after prolonged incubation in NO⁻₃ medium. It is suggested that NO⁻₃-metabolism in heterocysts is impaired due to a selective and irreversible loss of NO⁻₃-uptake and reductase systems resulting in the abolition of competition for molybdenum cofactor (Mo-Co) and reductant between nitrogenase and NO⁻₃-reductase, and an increase in glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase levels.     

Ammonium rhythm in cultures of the cyanobacterium Microcystis firma

Over a period of several days, rhythmic changes in extracellular NH⁺₄ concentration take place in cultures of the cyanobacterium Microcystis firma (Bré et Lenorm.) Schmidle, strain Gromov/St. Petersb. 398, under conditions of restricted CO₂ supply and light/dark alternation. The changes are enhanced by nitrate supply. Among the various processes generating intracellular NH⁺₄ (NH⁴₄ uptake, NO⁻₃ reduction, protein and amino acid degradation, photorespiration), NO⁻₃ reduction appears as the one most important. This can be concluded from experiments with and without nitrate and/or ammonium in the medium. In the presence of saturating CO₂, continuous light, or continuous darkness, rhythmic NH⁺⁴₄ oscillations are not induced. Studies of the incorporation of NH⁺₄ nitrogen by in vivo ¹⁵N-NMR show that if CO₂ is supplied, ¹⁵N is accumulated in several components with the following time course: in the first hour in Gln (δ), in the second hour in the α-amino groups of most nonbranched amino acids, in the third hour in γ-aminobutyric acid (GABA), Orn (δ) and Lys (ε), and in the sixth hour in Ala. Carbon limitation, however, results in accumulation of label in the amide nitrogen of glutamine only.     

Regulation of nitrate uptake into Chara corallina cells via NH+4 stimulation of NO−3 efflux

Abstract. Net NO₃ uptake by NO⁻₃ deficient Chara cells was used to calculate [NO⁻₃]_c assuming that the cytoplasm occupies 10% total volume and that nitrate reduction and storage are negligible (i.e. maximum [NO⁻₃]_c was calculated). A linear relationship was found between NO⁻₃ efflux and [NO⁻₃]_c. There was an initial burst of NO⁻₃ efflux when NH⁺₄ was added, followed by a slower efflux rate which matched influx rate such that net NO⁻₃ uptake was zero. Over 50% of NO⁻₃ that had been taken up in 2 h was lost within the first 5 min of NH⁺₄ addition. The Nernst equation was used to predict the direction of the electrochemical driving force for NO⁻₃ entry. Under the experimental conditions used NO⁻₃ efflux is actively transported. The differential involvement of both NO⁻₃ influx and NO⁻₃ efflux in the regulation of NO⁻₃ uptake is discussed and a model is proposed to account for these results which envisages discrete NO⁻₃ influx and NO⁻₃ efflux carriers.     

Nitrite in the root zone and its effects on ion uptake and growth of wheat seedlings

The immediate and posteffects of various concentrations of NaNO₂ on ion uptake of wheat ( Triticum aestivum L. cv. GK Öthalom) seedlings were studied at different pH values. Without pretreatment, the higher the concentration of NaNO₂ the greater was the decrease in uptake of K⁺ into the roots, both at pH 4 and pH 6. At pH 6 but not at pH 4 the reverse was true when the seedlings were pretreated with NaNO₂. Due to the high Na⁺ content of the roots, an effect of Na⁺ in this process cannot be excluded. Nitrite was taken up by the roots more rapidly than nitrate. Nitrite at 0.1 m M in the medium induced the development of an uptake system for both NO⁻₂ and NO⁻₃ in wheat roots. At higher concentrations pretreatment with NO⁻₂ decreased NO⁻₃ uptake by the roots, but NO₃ did not inhibit the uptake of NO₂. The toxic effect of NO⁻₂ was strongly pH dependent. Lower pH of the external solution led to an increased inhibition by NO⁻₂ of both ion uptake and growth of seedlings. The inhibitory effect of NO⁻₂ differed considerably for roots and shoots. The roots and especially the root hairs were particularly sensitive to NO⁻₂ treatment.     

Nitrate and nitrite reduction by alfalfa root nodules: Accumulation of nitrite in Rhizobium melioti bacteroids and senescence of nodules

Addition of NO⁻₃ rapidly induced senescence of root nodules in alfalfa ( Medicago sativa L. cv. Aragon). Loss of nodule dry matter began at the lowest NO⁻₃ concentration (10 m M ) but degradation of bacteroid proteins was only detected when nodules were supplied with NO⁻₃ concentrations above 20 m M .
Bacteroids from Rhizobium meliloti contained high specific activities of nitrate reductase (NR) and nitrite reductase (NiR). Both enzymes were presumably substrate-induced although substantial enzyme activities were present in the absence of NO⁻₃ Typical specific activities for soluble NR and NiR of bacteroids under NO⁻₃ free conditions were 1.2 and 1.4 μmol (mg protein)⁻¹h⁻¹, respectively. In the presence of NO⁻₃, the specific activity of NR was considerably greater than that of NiR, thus causing NO⁻₂ accumulation in bacteroids. Nitrite levels in the bacteroids were linearly correlated with specific activities of NR and NiR, indicating that NO⁻₂ is formed by bacteroid NR and that this NO⁻₂ in turn, induces bacteroid NiR. Accumulation of NO⁻₂ within bacteroids also indicates that NO⁻₂ inhibits nodule activity after feeding plants with NO⁻₃     

Effects of nitrogen source on betacyanin accumulation and growth in suspension cultures of Phytolacca americana

In suspension cultures of Phytolacca americana L., betacyanin accumulation per cell increased with increasing total nitrogen concentration (initial NH⁺₄:NO⁻₃ ratio 1:2) in the range 0–40 m M and then remained almost constant in the range 40–80 m M . Increasing ammonium increased growth while betacyanin accumulation was reduced. On the other hand, betacyanin accumulation increased when nitrate was increased while growth was almost constant in the concentration range examined. A time-course study of ammonium and nitrate concentration changes in the medium showed that betacyanin accumulation was associated with nitrate uptake.     

Induction of ammonium assimilation: leguminous roots compared with nodules using a split root system

A split root system for nitrogen uptake, in which one part of the root system was exposed to nitrogen-free nutrient and the other to circulated buffered ammonium, was used to investigate the effects of ammonium per se on the enzyme pathway for its assimilation in nodules and roots of leguminous plants. Plants of Trifolium repens L. cv. Grasslands Huia grown in the system showed similar growth and similar free amino acid content in the NH⁺₄-fed roots and in nodulated plants. Studies of ammonium assimilation using [¹³N]-NH⁺₄, applied to Glycine max [L.] Merr. cv. Amsoy plants, showed the label to be assimilated into amino acids in the NH⁺₄-fed roots and to be transported to the tops before subsequently appearing in the minus-N side of the split root system. Analysis of the xylem sap showed [¹³N]-asparagine to be the principal labelled amino acid component. In these plants, levels of both allantoate and the nodule-specific isoenzyme aspartate aminotransferase-P₂ were at least 10 times higher in the NH⁺₄-fed roots than in the minus-N side of the split root system. These studies strongly suggest that a nodule-type of ammonium assimilation was occurring in the NH⁺₄-fed side of the split root, and that this part of the root was transporting assimilatory products to the tops of the plants in a fashion analogous to that of a nitrogen-fixing nodule. These data implicate the involvement of NH⁺₄ in the induction of its own assimilatory pathway.     

Nitrate-induced de novo synthesis and regulation of NAD(P)H nitrate reductase from Sphagnum

The NO⁻₃-triggered induction of nitrate reductase (NR; EC 1.6.6.2) in the bryophyte Sphagnum magellanicum Brid. has been studied, using in vivo and in vitro assays as well as immunological methods. The time-course of induction was triphasic with maximal NR activity after 6–8 h. Results obtained from Western blots show that NR is synthesized de novo after NO⁻₃ application. The inhibitory effect of cycloheximide on NR induction corroborated this conclusion. Light enhanced the NO⁻₃-triggered NR induction. The enzyme activity, measured in vivo, increased more than the in vitro activity. No evidence for phytochrome control of NR was found. Nitrate uptake, in contrast to NR activity, showed no lag period after NO⁻₃ application and, under the experimental conditions used, was not rate limiting for NR induction. Neither light nor a NO⁻₃ pretreatment significantly affected NO⁻₃ uptake.     

Influence of nitrate and ammonium on the growth and 2,4-diaminobutyric acid composition of flatpea (Lathyrus sylvestris L.)

Abstract. Presence of 2.4-diaminobutyric acid (A₂bu), a neurotoxin, in tissues of flatpea ( Lathyrus sylvestris L.) necessitates a thorough understanding of the regulation of this nonprotein amino acid before the species can be recommended to livestock producers for forage applications. To determine how different concentrations and ratios of NO₃ and NH⁺₄ in growth media influence the levels of A₂bu and other free amino acids in the 'Lathco'flatpea cultivar, plants were grown hydroponically in controlled environments. The concentration of A₂bu was highest in tissues when the NO₃ to NH⁺₄ ratio in the nutrient solution was low. Responses of amides and other nonprotein amino acids, especially in the roots, followed a similar trend. Free protein amino acids in leaves and stems were generally unaffected by changes in NO₃ to NH⁺₄ ratios. In roots, protein amino acids increased as the NO₃ to NH⁺₄ ratio in the growth medium increased. Ammonium inhibited shoot and root growth; NO₃ alleviated the toxic effects of NH⁺₄. Soluble protein concentrations were higher in the shoots of NO₃-fed plants and in the roots of plants supplied with NH⁺₄. These results suggest that accumulation of A₂bu and other nonprotein amino acids, as well as asparagine and glutamine, plays a role in detoxification of NH⁺₄ and storage of N.     

Induction of nitrate reductase in needles of Scots pine seedlings by NOX and NO3−

The possibility to induce nitrate reductase (NR; EC 1.6.6.2) in needles of Scots pine ( Pinus sylvestris L.) seedlings was studied. The NR activity was measured by an in vivo assay. Although increased NR activities were found in the roots after application of NO₃⁻, no such increase could be detected in the needles. Detached seedlings placed in NO₃⁻ solution showed increasing NR activities with increasing NO₃⁻ concentrations. Exposure of seedlings to NO_x (70–80 ppb NO₂ and 8–12ppb NO) resulted in an increase of the NR activity from 10–20 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹ to about 400 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. This level was reached after 2–4 days of exposure, thereafter the NR activity decreased to about 200 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. Analyses of free amino acids showed low concentrations of arginine and glutamine in NO_x-fumigated seedlings compared to corresponding controls.     

Simulation of nitrogen assimilation by heterotrophic soil microbial biomass

Assimilation of N by heterotrophic soil microbial biomass is associated with decomposition of organic matter in the soil. The form of N assimilated can be either low molecular weight organic N released from the breakdown of organic matter (direct assimilation), or NH⁺₄ and NO⁻₃ from the soil inorganic N pool, into which mineralized organic N is released (mineralization immobilization turnover). The kinetics of C and N turnover in soil is quantifiable by means of computer simulation models. NCSOIL was constructed to represent the two assimilation schemes. The rate of N assimilation depends on the rate of C assimilation and microbial C/N ratio, thereby rendering it independent of the assimilation scheme. However, if any of the N forms is labeled, a different amount of labeled N assimilation will be simulated by the different schemes. Experimental data on inorganic N and ¹⁵N and on organic ¹⁵N dynamics in soils incubated with ¹⁵N added as NH⁺₄ or organic N were compared with data simulated by different model schemes. Direct assimilation could not account for the amount of ¹⁵N assimilated in any of the experimental treatments. The best fit of the model to experimental data was obtained for the mineralization immobilization turnover scheme when both NH⁺₄ and NO⁻₃ were assimilated, in proportion to their concentration in the soil.     

Nitrate reductase and molybdenum cofactor in annual ryegrass as affected by salinity and nitrogen source

The influence of salinity on the activity of nitrate reductase (NR, EC 1.6.6.1) and the level of the molybdenum cofactor (MoCo) as affected by the source and concentration of nitrogen was studied in annual ryegrass ( Lolium multiflorum cv. Westerwoldicum). Plants grown in sand were irrigated with nutrient solution with an electrical conductivity of 2 or 11.2 dS m⁻¹, containing nitrogen (0.5 or 4.5 m M ) in the form of NH₄NO₃ or NaNO₃ Salinity-treated (11.2 dS m⁻¹) plants produced less biomass and more organic nitrogen while accumulating more NO⁻₃ than control plants. Increased nitrogen concentration in the irrigation solutions enhanced biomass and organic nitrogen production as well as NO⁻₃ accumulation irrespective of the electrical conductivity. Salinity inhibited shoot growth and increased shoot NR activity of plants receiving 4.5 m M NH₄NO₃ or NaNO₃. Similar effects were observed in roots of plants grown in 4.5 m M NaNO₃. Nitrate added to a complementation medium containing ryegrass MoCo and the NR apoprotein of Neurospora crassa mutant nit-1 stimulated the activity of the reconstituted NR (NADPH-nitrate reductase, EC 1.6.6.3). Increased salinity and nitrogen in the nutrient solutions caused an increase of MoCo content in roots and shoots. Similar results were observed for NR activity in the shoots. The increase of MoCo in response to salinity was more pronounced than that of NR, especially in the roots. We conclude that the pool size of MoCo in ryegrass is not constant, but varies in response to nutritional and environmental factors.     

Culture of mycorrhizal tree seedlings under controlled conditions: Effects of nitrogen and aluminium

A sand culture system was developed for growth of mycorrhizal seedlings under monoxenic conditions, with frequently renewed nutrient solution The composition of the nutrient solution resembled that of a forest soil solution, based on long-term measurements from forest sites at Soiling, northern Germany. Seedlings of Picea abies (L.) Karst. inoculated with Lactarius rufus (Scop.) Fr. were grown in this culture system. Plants developed rapidly, having almost totally mycorrhizal root systems. Nitrate at 2.7 mM in the nutrient solution and applied over a 13 week period had no negative effect on mycorrhizal development. Ammonium at 2.7 mM reduced the degree of mycorrhizal infection slightly, in such a way that the degree of mycorrhizal infection was reduced to a much less extent than the total number of root lips. Hence, the impact of NH⁺₄ may be primarily on root development and not on mycorrhizal fungal colonization. When the concentrations of NO-₃ and NH⁺₄ used in the present study are compared to those found in forest soil solutions, NO-₃ and NH₄⁺ would not appear to influence mycorrhizal development negatively under natural conditions. Aluminium at 0.8 mM and applied over a 13 week period reduced Mg uptake into roots and needles by 52 and 64%, respectively, resulting in needle chlorosis and strongly reduced photosynthetic activity. From a comparison of this study with others, no major difference in physiological response to aluminium exposure between non-mycorrhizal seedlings and seedlings colonized with Lactarius rufus was found.     

Allantoinase and asparaginase activities in maturing fruits of nodulated and non -nodulated soybeans

Immature fruits of soybean ( Glycine max L. Merr. cv. Santa Rosa) were found to contain high ureide/amino acid ratios for plants dependent on atmospheric nitrogen (nodulated), but low ratios for plants cultivated on NO⁻₃ (non-nodulated). The pod tissue was responsible for almost all this difference, which reflects the N metabolism of these plants (nodulated:urcide-based; NO⁻₃ dependent: asparagine based). The capacity of fruit tissues to utilize ureides and asparagine via allantoinase (EC 3.5.2.5) and asparaginase (EC 3.5.1.1) was investigated during fruit development. Both enzymes were present in crude desalted extracts of all parts of the fruit analysed (pod, cotyledon and seed coat). Asparaginase was detected in pod tissue only at early stages and with very low activities, whereas high activities of allantoinase (up to 20 [imol pod⁻¹ h⁻¹) were present after this organ reached full expansion. The cotyledons contained most of the allantoinase and asparaginase activities of the seed, the highest activities being recorded during the period of rapid protein accumulation. There was little difference in the activity patterns for nodulated and NO⁻₃-grown plants, despite the large difference in nitrogen nutrition of the fruits.     

Low external pH prevents cell elongation but not multiplication of embryogenic carrot cells

Embryogenic cultures of cultivated carrot ( Daucus crota cv. Scarlet Nantes) were initiated from seedling hypocotyls on hormone-containing nutrient medium and from wounded zygotic embryos on hormone-free medium. Both of these cultures were maintained with continuous multiplication as unorganized, embryogenic cell masses on hormone-free medium at pH 4.0, containing NH⁺₄ as the sole nitrogen source. When grown on hormone-free medium at pH 4.0, neither culture contained any elongated cells. Virtually all cells were densely cytoplasmic and nearly spherical. Some cells were enlarged, not densely cytoplasmic, but always spherical. When either culture was transferred to an auxin-containing medium at pH 5.8, numerous elongated cells were produced. Elongated cells were observed when either naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid was used, and whether the nitrogen source was NH⁺₄ alone or a combination of NH⁺₄ and NO⁻₃. Elongated cells were more abundant when a combined nitrogen source was used. When cultures containing elongated cells were transferred to and multiplied on hormone-free or hormone-containing medium buffered at pH 4.0, all elongated cells disappeared after 2 weeks. No elongated cells were observed in any of the lines tested at pH 4.0. These results clearly show that it was the pH of the culture medium and not the presence or absence of an auxin or the nitrogen source(s) that permitted or prevented cell elongation in the embryogenic cultures tested.