Response of nitrogen metabolism to boron toxicity in tomato plants

Boron (B) toxicity has become important in areas close to the Mediterranean Sea where intensive agriculture has been developed. The objective of this research was to study the effects of B toxicity (0.5 m m and 2.0 m m B) on nitrogen (N) assimilation of two tomato cultivars that are often used in these areas. Leaf biomass, relative leaf growth rate (RGR_L), concentration of B, nitrate (NO₃⁻), ammonium (NH₄⁺), organic N, amino acids and soluble proteins, as well as nitrate reductase (NR), nitrite reductase (NiR), glutamine synthase (GS), glutamate synthetase (GOGAT) and glutamate dehydrogenase (GDH) activities were analysed in leaves. Boron toxicity significantly decreased leaf biomass, RGR_L, organic N, soluble proteins, and NR and NiR activities. The lowest NO₃⁻ and NH₄⁺ concentration in leaves was recorded when plants were supplied with 2.0 m m B in the root medium. Total B, amino acids, activities of GS, GOGAT and GDH increased under B toxicity. Data from the present study prove that B toxicity causes inhibition of NO₃⁻ reduction and increases NH₄⁺ assimilation in tomato plants.     

Osmoregulation and role of nitrate during regrowth after cutting of ryegrass (Lolium perenne)

The osmotic role of nitrate during aftermath growth of Lolium perenne L. cv. Réveille was investigated. Plants were grown from seed in a controlled environment using a liquid medium with 1.0 m M NH₄NO₃ as nitrogen source.
Eight-week-old plants were cut 4.0 cm above the root system and then harvested over a 14-day period of regrowth on the same initial nutrient solution, except that nitrate was ¹⁵N labelled. Throughout the experimental period, nitrate storage and reduction in roots were low. In stubble and especially in leaves, nitrate accumulated during the first 6 days of regrowth whereas nitrate reduction mainly occurred after this period. Analyses of carbohydrate, chloride and potassium contents in stubble and leaves showed that the accumulation of nitrate osmotically compensated for the decrease in soluble sugars during the first 6 days of regrowth.
The cumulative osmotic potential of sugars, chloride and nitrate in differently treated plants was studied in stubble and leaves. Compared with uncut plants, the lower carbohydrate concentrations found in cut plants regrowing on 1.0 m M NH₄NO₃ were compensated for by an accumulation of nitrate. During aftermath growth on low nitrogen nutrition (0.2 m M NH₄NO₃), chloride replaced nitrate, supporting the proposed osmotic function of nitrate.
It is concluded that nitrate is involved in the osmotic adjustment of ryegrass during regrowth after cutting.     

Sink control of nitrogen uptake and assimilation during regrowth after-cutting of ryegrass (Lolium perenne L.)

Abstract. The ¹⁵N isotope was used to compare the uptake and the assimilation of NH₄⁺ and NO₃ nitrogen in ryegrass ( Lolium perenne L.) during regrowth after cutting. Uptake of nitrate-N, expressed per plant, was at all times greater than ammonium-N uptake and assimilation decreased in roots and stubble while its assimilation was maintained at a high level in leaves. It has been suggested that ammonium assimilation is directly related to the availability of carbohydrates in the sink organ (leaves) resulting from their remobilization from the source organs (roots and stubble). Nitrate reduction decreased in all organs, while the uptake of NO₃ was still high. After this first period of regrowth, nitrogen assimilation both from nitrate and ammonium increased in all the plants. Nitrate reduction capacity (expressed in μg NO₃-N reduced per g D.W. per d) is 7.5 and 22.5 times greater in leaves than in stubble and roots, respectively. Therefore, nitrogen assimilation in stubble and particularly in roots was mainly dependent on ammonium nitrogen.     

Effect of darkness and CO2 starvation on NH4+ and NO3− assimilation in the unicellular alga Cyanidium caldarium

Cyanidium caldarium (Tilden) Geitler, a non-vacuolate unicellular alga, resuspended in medium flushed with air enriched with 5% CO₂, assimilated NH₄⁺ at high rates both in the light and in the dark. The assimilation of NO₃⁻, by contrast, was inhibited by 63% in the dark. In cell suspensions flushed with CO₂-free air, NH₄⁺ assimilation decreased with time both in the light and in the dark and ceased almost completely after 90 min. The addition of CO₂ completely restored the capacity of the alga to assimilate NH₄⁺. NO₃⁻ assimilation, by contrast, was 33% higher in the absence of CO₂ and was linear with time. It is suggested that NO₃⁻ and NH₄⁺ metabolism in C. caldarium are differently controlled in response to the light and carbon conditions of the cell.     

Increase in glutamate synthase (NADH) activity in maize seedlings in response to nitrate and ammonium nitrogen

Roots and leaves of Zea mays L. cv. Ganga Safed-2 seedlings grown with nutrient solution containing either 10 m M KNO₃ or NH₄Cl or 5 m M NH₄NO₃ had considerably higher glutamate synthase (NADH, EC 1.4.1.14) activity than the corresponding organs from seedlings grown without any nitrogen. The supply of inorganic nitrogen for a short time, i.e. 3 h, to roots and leaves excised from seedlings grown without nitrogen also increased the enzyme activity in these organs. This increase was more pronounced with nitrate than with ammonium nitrogen. When excised roots and leaves from NH₄NO₃-grown seedlings were incubated in a minus nitrogen medium for 24 h, the enzyme activity declined considerably. This decline was inhibited to some extent by nitrogen, especially by nitrate. Inorganic nitrogen prevented similarly the decline in in vitro enzyme activity during 24 h storage at 25°C, more regularly for the root than for the leaf enzyme. The experiments demonstrate the role of inorganic nitrogen in the regulation of glutamate synthase activity.     

Varying the ratio of 15N-labelled ammonium and nitrate-N supplied to creeping bent: effects on nitrogen absorption and assimilation, and plant growth

The relative rates of ammonium and nitrate-N uptake and assimilation by creeping bent ( Agrostis stolonifera ), were investigated for plants grown in soil and supplied with three different ratios of ammonium and nitrate-N. Following two preliminary defoliations, plants were supplied with the equivalent of 150 kg N ha⁻¹, given as ¹⁵N-(differentially) labelled NH₄⁺ and NO₃⁻-N in three different ratios (20:80, 50:50 and 80:20), followed by sequential destructive harvests of shoots and roots at four points during a 35-d regrowth period. Maximum use of labelled nitrogen and 'exhaustion' of soil mineral nitrogen reserves occurred much earlier when plants were supplied with half or more of their nitrogen as ammonium, than occurred when they were supplied predominately with nitrate-N. The lack of consistency in the patterns of ammonium and nitrate-N absorption, however, implied that the plants had no specific preference for either nitrogen form. Supplying plants with different combinations of ammonium and nitrate produced distinctive differences in plant morphology. In the high nitrate treatment, plants preferentially partitioned resources into shoot and stolon formation, whereas in the high ammonium treatment, resources were preferentially partitioned into root production. These changes in plant morphology might be adaptations to aid species survival in environments associated with a predominance of either nitrogen form.     

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.     

Nitrogen regulation of erythromycin formation in Streptomyces erythreus

Abstract Erythromycin formation decreased in Streptomyces erythreus as a function of the ammonium concentration present in the medium. Total inhibition of synthesis was obtained with 100 mM NH₄Cl but medium pH and culture growth were not significantly affected. A similar effect was obtained with NH₄NO₃ or (NH₄)₂SO₄ indicating that ammonium ion probably repressed formation of antibiotic.     

Characteristics of ammonium absorption by excised root and leaf tissues of maize

Absorption of ammonium from solutions of ammonium chloride by maize ( Zea mays L. cv. GS-2) tissue was studied. In contrast to an initial rapid phase of absorption in root tissue, a one hour lag period was recorded in leaf tissue. The maximum rate of uptake was observed at 5–10 m M NH₄Cl in both tissues. Roots had a K_m value of 1.0 m M and V_max of 24.3 μmol ammonium (g fresh weight)⁻¹ h⁻¹, whereas the leaf tissue had a higher K_m (4.1 m M ) and a lower V_max (8.7 μmol). There was a concentration dependent increase in ethanol soluble and insoluble fractions of organic nitrogen during ammonium supply. The optimum pH for ammonium absorption for both tissues was 7.4. The optimal concentration of CaCl₂ for ammonium absorption was 5 m M whereas that of KCl was only 1 m M . In both tissues, the absorption was inhibited substantially by DCMU, DNP, cycloheximide, lincomycin, sodium tungstate, sodium arsenate and to some extent also by the anions nitrate and sulfate. It is suggested that a carrier is involved in an active uptake of ammonium in the leaf tissues.     

Nitrogen assimilation in Lolium perenne colonized by the arbuscular mycorrhizal fungus Glomus fasciculatum

To investigate nitrogen assimilation in Lolium perenne L. colonized by the arbuscular mycorrhizal (AM) fungus Glomus fasciculatum (Thax. sensu Gerd.), nitrate uptake, key enzyme activities, and ¹⁵N incorporation into free amino acids were measured. After a 4-h labelling period with [¹⁵N]nitrate, ¹⁵N content was higher in roots and shoots of AM-plants than in those of control plants. Glutamine synthetase (GS) and nitrate reductase (NR) activities were increased in shoots of AM-plants, but not in roots. More label was incorporated into amino acids in shoots of AM plants. Glutamine, glutamate, alanine and γ-aminobutyric acid were the major sinks for ¹⁵N in roots and shoots of control and AM plants. Interactions between mycorrhizal colonization, phosphate and nitrate nutrition and NR activity were investigated in plants which received different amounts of phosphate or nitrate. In shoots of control plants, NR activity was not stimulated by high levels of phosphate nutrition but was stimulated by high levels of nitrate. At 4 m M nitrate in the nutrient solution, NR activity was similar in control and AM plants. We concluded that mycorrhizal effects on nitrate assimilation are not mediated via improved phosphate nutrition, but could be due to improved nitrogen uptake and translocation.     

Effect of inhibitors on ammonium assimilation in Chlorella sorokiniana in light and darkness

In N-sufficient cells of Chlorella sorokiniana Shihira and Krauss strain 211/8K (CCAP of Cambridge University), assimilation of ammonium was strictly dependent on light and CO₂, and was severely inhibited by 100 μ M atrazine or 10 μ M 3-(3,4-dichlorophenyl)-1, l-dimethylurea (DCMU). In N-limited cells, assimilation of NH₄⁺ took place at similar rates in both light and darkness, which were 1.6-fold higher than the rate of light-dependent assimilation by N-sufficient cells. Assimilation by N-limited cells was inhibited by l -methionine- dl -sulfoximine (MSX), but not by atrazine or DCMU.
The rate of photosynthetic O₂ evolution was 2.9±0.9 mmol ml⁻¹ packed cell volume (PCV) h⁻¹ in N-sufficient cells, and 0.64±0.12 mmol ml⁻¹ PCV h⁻¹ in N-limited cells. In the latter resupply of ammonium resulted in a rapid activation by 22%;, followed by a time-dependent increase of the photosynthetic O₂ evolution, which after 12 h reached the same rate as in N-sufficient cells.
Respiratory consumption of oxygen in darkness in N-sufficient and N-limited cells was 0.10±0.03 and 0.11±0.02 mmol ml⁻¹ PCV h⁻¹, respectively. Addition of ammonium was without effect on respiration of N-sufficient cells, but resulted in a 4-fold stimulation of respiration of N-limited cells. Such stimulation took place also in cells treated with DCMU, atrazine, or MSX, and it was also promoted by methylammonium. The stimulation of respiration lasted for several hours.     

KINETICS OF AMMONIUM ASSIMILATION IN TWO SEAWEEDS, ENTEROMORPHA SP. (CHLOROPHYCEAE) AND OSMUNDARIA COLENSOI (RHODOPHYCEAE)

The kinetics of ammonium assimilation were investigated in two seaweeds from northeastern New Zealand, Enteromorpha sp. (Chlorophyceae, Ulvales) and Osmundaria colensoi (Hook. f. et Harvey) R.E. Norris (Rhodophyceae, Ceramiales), with the use of a recently developed method for measuring assimilation. In contrast to ammonium uptake, which was nonsaturable, ammonium assimilation exhibited Michaelis–Menten kinetics in both species. Maximum rates of assimilation (V_max) were 27 and 12 μmol·(g DW)⁻¹·h⁻¹ for Enteromorpha sp. and O. colensoi, respectively, with half-saturation (K_m) constants for assimilation of 18 and 41 μM. At environmentally relevant concentrations, assimilation accounted for all of the ammonium taken up by both species. The maximum rate of assimilation in Enteromorpha sp. resembled very closely that of the ammonium assimilatory enzyme, glutamine synthetase, when activities of the latter were measured in the presence of subsaturating substrate (glutamate and ATP) concentrations. Moreover, the initial rate of glutamine production (measured with HPLC) following ammonium enrichment was almost identical to the rates determined above. The rate of ammonium assimilation was therefore determined by three independent methods, two of which involve in vivo measurements, and it is suggested that the use of assimilation kinetics may be useful when examining the nutrient relations of seaweeds.     

Isolation and characterization of nitrate reductase deficient mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum

To clarify the role of the fungal nitrate assimilation pathway in nitrate reduction by mycorrhizal plants, nitrate reductase (NR)-deficient (NR⁻) mutants of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum Romagnesi have been selected. These mutants were produced by u.v. mutagenesis on protoplasts originating from homokaryotic mycelia belonging to complementary mating types of this heterothallic tetrapolar species. Chlorate-resistant mutants were first selected in the presence of different nitrogen (N) sources in the culture medium. Among 1495 chlorate resistant mycelia, 30 failed to grow on nitrate and lacked a detectable NR activity. Growth tests on different N sources suggested that the NR activity of all the different mutants is specifically impaired as a result of mutations in either the gene coding for NR apoprotein or genes controlling the synthesis of the molybdenum cofactor. Furthermore, restoration of NR activity in some of the dikaryons obtained after crosses between the different mutant mycelia suggested that not all the selected mutations mapped in the same gene. Utilization of N on a NH₄¹⁵NO₃ medium was studied for two mutant strains and their corresponding wild-type homokaryons. None of the mutants could use nitrate whereas ¹⁵N enrichment values indicated that 13–27% of N present in 13-d-old wild-type mycelia originated from nitrate. Apparently, the mutant mycelia do not compensate their inability to use nitrate by a more efficient use of ammonium. These different NR mutants still form mycorrhizas with the habitual host plant, Pinus pinaster (Ait.), making them suitable for study of the contribution of the fungal nitrate assimilation pathway to nitrate assimilation by mycorrhizal plants.     

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.     

Photosynthate costs associated with the utilization of different nitrogen–forms: influence on the carbon balance of plants and shoot–root biomass partitioning

The photosynthate costs of processes (amino acid and protein synthesis and turnover, and pH regulation) associated with the utilization of nitrate (NO₃⁻), ammonium (NH₄⁺) or glutamine (Gln) for plant growth were estimated. Based on these estimates, the effects of these forms of nitrogen (N) on the carbon balance of plants and on shoot–root biomass allocation were evaluated. The results indicated that NO₃⁻ as an N source for plant growth is not substantially more expensive to utilize than either NH₄⁺ or Gln, particularly in the long term when costs due to protein turnover dominate the total costs of N utilization. It is also suggested that the photosynthate use in processes associated with N assimilation has little impact on the carbon balance of plants, and hence on shoot–root biomass allocation.     

Effects of glutamine and K-glutamate on assimilation of [15N]-nitrate during auxin treatment for root formation in vitro (Pinus pinaster)

In order to establish the relationship of nitrogen nutrition to root formation in vitro, efficiency of various forms of nitrogen supply was investigated in micropropagated shoots of Pinus pinaster. Nitrate (3.3 m M as N) containing medium supplemented with glutamine (2 m M as N) resulted in optimal rooting (control medium) whereas the rooting response was inhibited by the presence of glutamate (2 m M as N) in the medium. Study of ¹⁵NO⁻₃ behaviour in shoots cultured on the control medium showed a marked increase of ¹⁵N incorporation during the second half of a period of auxin treatment (total duration 16 days). The majority of the nitrogen incorporated from the medium was accumulated into protein in the rooting zone. Autoradiography of [³H]-thymidine in this reacting zone showed strong DNA synthesis at this time. Addition of glutamate both prevented the root formation process and resulted in a marked slowdown of nitrate uptake and decrease of protein synthesis.     

Molecular approaches to the analysis of nitrate assimilation

Abstract. The application of molecular approaches such as mutant analysis and recombinant DNA technology, in conjunction with immunology, are set to revolutionize our understanding of the nitrate assimilation pathway. Mutant analysis has already led to the identification of genetic loci encoding a functional nitrate reduction step and is expected to lead ultimately to the identification of genes encoding nitrate uptake and nitrite reduction. Of particular significance would be identification of genes whose products contribute to regulatory networks controlling nitrogen metabolism. Recombinant DNA techniques are particularly powerful and have already allowed the molecular cloning of the genes encoding the apoprotein of nitrate reductase and nitrite reductase. These successes allow for the first lime the possibility to study directly the role of environmental factors such as type of nitrogen source (NO₃⁻ or NH₄⁺) available to the plant, light, temperature water potential and CO₂ and O₂ tensions on nitrate assimilation gene expression and its regulation at the molecular level. This is an important advance since our current understanding of the regulation of nitrate assimilation is based largely on changes of activity of the component steps. The availability of mutants, cloned genes, and gene transfer systems will permit attempts to manipulate the nitrate assimilation pathway.     

ASSAY OPTIMIZATION AND REGULATION OF UREASE ACTIVITY IN TWO MARINE DIATOMS

An in vitro urease enzyme assay was developed for the marine diatoms Thalassiosira pseudonana Hasle et Heimdal (clone 3H) and T. weissflogii (Grunow) Fryxell et Hasle (clone Actin). This assay involves the colorimetric measurement of ammonium following the hydrolysis of urea in crude cell homogenates and it is the first assay to account for the rate of nitrogen assimilation in both species grown on urea as the sole nitrogen source. Urease activity was found to be present regardless of nitrogen source, although activities showed distinctly different patterns depending on the species examined and form of nitrogen supplied. Under nitrogen-replete conditions, urease activity in T. pseudonana was present constitutively when grown on NH₄⁺ and upregulated when grown on NO₃⁻ or urea. In nitrogen-replete T. weissflogii , urease activity was present at high constitutive levels regardless of the nitrogen source and showed no upregulation. Nitrogen starvation did not upregulate activity in either species.     

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.     

Carbon and nitrogen partitioning in young nodulated pea (wild type and nitrate reductase-deficient mutant) plants exposed to NH4NO3

Carbon and nitrogen partitioning was examined in a wild-type and a nitrate reductase-deficient mutant (A317) of Pisum sativum L. (ev. Juneau), effectively inoculated with two strains of Rhizobium leguminosarum (128C23 and 128C54) and grown hydroponically in medium without nitrogen for 21 days, followed by a further 7 days in medium without and with 5 mM NH₄NO₃. In wild-type symbioses the application of NH₄NO₃ significantly reduced nodule growth, nitrogenase (EC 1.7.99.2) activity, nodule carbohydrates (soluble sugars and starch) and allocation of [¹⁴C]-labelled (NO₃⁻, NH₄⁺, amino acids) in roots. In nodules, there was a decline in amino acids together with an increase in inorganic nitrogen concentration. In contrast, symbioses involving A317 exhibited no change in nitrogenase activity or nodule carbohydrates, and the concentrations of all nitrogenous solutes measured (including asparagine) in roots and nodules were enhanced. Photosynthate allocation to the nodule was reduced in the 128C23 symbiosis. Nitrite accumulation was not detected in any case. These data cannot be wholly explained by either the carbohydrate deprivation hypothesis or the nitrite hypothesis for the inhibition of symbiotic nitrogen fixation by combined nitrogen. Our result with A317 also provided evidence against the hypothesis that NO₃⁻ and NH₄⁺ or its assimilation products exert a direct effect on nitrogenase activity. It is concluded that more than one legume host and Rhizobium strain must be studied before generalizations about Rhizobium /legume interactions are made.