Assimilation of nitrate and ammonium by the Scots pine (Pinus sylvestris) seedling under conditions of high nitrogen supply

Seedlings of Scots pine ( Pinus sylvestris L.) were grown on perlite for 21 days under controlled conditions. Apart from the water control, KNO₃ (15 m M ), (NH₄)₂SO₄ (7.5 m M ), and NH₄NO₃ (15 m M ) were offered to study the effects of a high nitrogen supply on nitrogen assimilation. In some experiments 1.3 m M potassium was added to the basic ammonium solutions. In labelling studies nitrate and ammonium were 2.3 atom%¹⁵N-enriched. It was found that over the 21-day period approximately three times more ammonium-N was taken up than nitrate-N. However, nitrate and ammonium, applied simultaneously, were taken up to the same extent as if they were applied separately (additivity). The presence of K⁺ in the medium did not affect N-uptake. Among the soluble N-containing compounds nitrate, ammonium and 8 amino acids were quantified. It was found that assimilation of nitrate can cope with the uptake of NO⁻₃ under all circumstances. Neither free nitrate nor ammonium or amino acids accumulated to an extent exceeding the values of water-grown seedlings. On the other hand, in case of high ammonium supply considerably more nitrogen was taken up than could be incorporated into nonsoluble N-containing substance ('protein'). The remaining nitrogen was found to accumulate in intermediary storage pools (free NH₄⁺, glutamine, asparagine, arginine). Part of this accumulated N could be incorporated into protein when potassium was offered in the nutrient solution. It is concluded that potassium is a requirement for a high rate of protein synthesis not only in crop plants but also in conifers.     

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.     

Net fluxes and pools of nitrogenous compounds during suspension culture of photoautotrophic Chenopodium rubrum cells

Abstract. Suspension cultured cells of Chenopodium rubrum were grown photoautotrophically under a diurnal light-dark cycle of 16-8h. The following phases of the batch culture were differentiated: a short lag, a cell division phase terminated by a pronounced transition to stationary maintenance which finally gradually passed into senescence. Nitrogen fluxes typical of these stages were followed by measuring uptake of NO₃ and NH₄⁺ from the medium and their incorporation into the cellular fractions of nitrogenous compounds. Activities of seven N-metabolizing enzymes were determined. Compartmentation of enzymes and nitrogenous compounds was analysed after isolation of intact chloroplasts and vacuoles from protoplasts. Eighty-two per cent of the N originally present in the medium was taken up and incorporated to an extent of 80% into protein until the end of the division phase. Net protein synthesis ceased upon transition to the stationary phase. During the division phase a vacuolar pool of NO₃ was established and then maintained throughout the resting phase. Free cellular NH₄⁺ was not localized within the vacuole and responded to the ammonium content of the medium. Amino acids accumulated in the cells especially during the stationary phase, during which they were present in the vacuole. Typical nitrogen relations are portrayed as flux diagrams for one day of each of the essential developmental phases. The enzyme activities were easily sufficient to account for the observed flow rates of the corresponding nitrogenous compounds. Hence, uptake of NO₃ and NH₄⁺ must be considered as steps limiting N metabolism in Chenopodium rubrum cell suspensions.     

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.     

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.     

Glycolate metabolism in cyanobacteria. III. Nitrogen controls excretion and metabolism of glycolate in Anabaena cylindrica

The effect of nitrogen on excretion and metabolism of glycolate in Anabaena cylindrica (CCAP 1403/2a) was studied. Glycidate, an inhibitor of glutamate:glyoxylate aminotransferase (EC 2.6.1.4), reduced the L-methionine-DL-sulfoximine-induced NH₄⁺ release by ca 40%, while net CO₂ fixation and C₂H₂ reduction were not lowered. This indicates that at least a part of the glyoxylate synthesized in A. cylindrica is metabolized via glycine to serine. Addition of NH₄Cl or glutamate to the medium reduced the excretion of glycolate. At pH 9, under air, NH₄Cl reduced the excretion by 10–30% and under high pO2 (0.03 kPa CO₂ in O₂) by about 80–90%. At pH 7.5, under high pO₂, NH₄Cl and glulamate reduced the excretion by about 40 and 80%, respectively. Also, the presence of NH₄Cl stimulated the animation of glyoxylate under such conditions as shown by an increased glycine pool and a decreased glutamate pool. We suggest that nitrogen regulates the capacity of A. cylindrica to retain and recycle glycolate intracellularly and that glutamate serves as an amino donor in the conversion of glyoxylate to glycine.     

Ammonium uptake by field-grown Eriophorum vaginatum roots under laboratory and simulated field conditions

Nitrogen (N) deficiencies in tundra ecosystems could be caused, in part, by the kinetics of root N uptake. The objectives of this study were to quantify NH₄ uptake by field-grown excised roots of Eriophorum vaginatum I. under controlled NH₄ concentrations (0-250 μmol I^-1) and temperatures (5-20°C) and to evaluate this laboratory derived model as a means of estimating field NH₄ uptake. There was no consistent temperature effect on root NH₄ uptake which suggests a relative in-sensitivity of E. vaginatum roots to short-term temperature fluctuations. The Michaelis-Menten equation parameters for NH₄ uptake were V_max= 22.1 μmol h^-1 g^-1 and K_m= 191 μmol I^-1. Using field NH₄ concentrations, field E. vaginatum root biomass data, and the Michaelis-Menten equation, an estimate was made of NH₄ uptake over a 42 day period; this estimate of NH₄ uptake accounted for 28% of the net incorporation of N into leaves and roots which is a reasonable estimate for E. vaginatum which relies primarily on N retranslocation for supplying new leaves and roots. Major uncertainties in field N uptake rates, model parameterization, and site characterization preclude an accurate model validation and indicate research areas most in need of future study.     

Glucose transport throughout fermentation by Saccharomyces cerevisiae NCYC 1108

The rate of fermentation of glucose by a polyploid strain of Saccharomyces cerevisiae growing in a defined salts medium depends on the availability of NH₄+⁺. Its decline after exhaustion of the nitrogen source corresponded with the ability of the cells to accumulate the glucose analogue 2-deoxyglucose. Addition of NH₄+⁺to a nitrogen-depleted culture stimulated both glucose utilization and 2-deoxyglucose uptake. Since stimulation was inhibited by cycloheximide, maintenance of glucose transport during fermentation is dependent on protein synthesis.     

SALT-DEPENDENT PROPERTIES OF HATCHING ENZYME FROM EMBRYOS OF THE SEA URCHIN, HEMICENTROTUS PULCHERRIMUS

The effect of salts on hatching enzyme and protease from the embryo of the sea urchin, Hemicentrotus pulcherrimus , was investigated. The culture medium containing hatching enzyme secreted from the hatched blastula was dialyzed against Tris-HCl (pH 8.0) with or without salts. Both hatching enzyme and protease were activated and stabilized by CaCL₂, NaCI and KCI, while inhibited by MgCI₂. Protease activity was maximal at about 0.25 M NaCI. KCI, NH₄, CI and LiCI. Maximal activity of hatching enzyme was obtained at 0.5 M NaCl, KCI and NH₄ CI, while activity was inhibited by any concentration of LiC1. Among monovalcnt cations, the order of activation was NaCI, KCI > NH₄Cl. The activity of hatching enzyme was stabilized by dialysis against 1 M NaCI or KCI in the presence of CaCl.₂, but was rapidly lost by dialysis against lower concentrations of salts. Reactivation of hatching enzyme was not achieved by redialysis against I M NaCI. On the other hand, protease was reactivated by I M NaCI or KCI. From these results, hatching enzyme of the sea urchin may be called a moderate halophilic enzyme. It was assumed that at least two enzymes exist in the crude enzyme preparation and that they may have different functions.     

Fixation of molecular nitrogen by Methanosarcina barkeri

Abstract Methanosarcina barkeri cells were observed in ammonia-free anaerobic acetate enrichments for sulfate-reducing bacteria. The capacity of Methanosarcina to grow diazotrophically was proved with a pure culture in mineral media with methanol. The cell yields with N₂ or NH₄⁺ ions as nitrogen source were 2.2 g and 6.1 g dry weight, respectively, per mol of methanol. Growth experiments with ¹⁵N₂ revealed that 84% of the cell nitrogen was derived from N₂. Acetylene was highly toxic to Methanosarcina and only reduced at concentrations lower than 100 μmol dissolved per 1 of medium. Assimilation of N₂ and reduction of acetylene were inhibited by NH₄⁺ ions. The experiments show that N₂ fixation occurs not only in eubacteria but also in archaebacteria. The ecological significance of diazotrophic growth of Methanosarcina is discussed.     

Mycobiont-cyanobiont interactions during dark nitrogen fixation by the lichen Peltigera aphthosa

Acetylene reduction (nitrogenase activity) by excised cephalodia of Peltigera aphthosa Willd. slowly declined on transfer of the cephalodia from light to darkness. The decline was more rapid in the absence of CO₂ or when phosphoenolpyruvate carboxylase activity was inhibited by adding maleic acid or malonic acid. When glutamine synthetase (GS) activity was totally inhibited by adding l -methionine- dl -sulphoximine (MSX) the decline in nitrogenase activity in the absence of CO₂ still occurred. However, this loss of activity did not occur when the mycobiont was disrupted using digitonin (0.01 % w/v) and the fixed NH₄⁺ was released into the medium. The data suggest that dark CO₂ fixation by the fungus supplies carbon skeletons which remove newly fixed NH₄⁺ produced by the cyanobacterium. When such carbon skeletons are not available MH₄⁺ accumulates and inhibits nitrogenase activity even in the absence of GS activity. It is probable that NH₄⁺ and a product of GS exert independent inhibitory effects on nitrogenase activity.     

A precise potentiometric method for determination of algal activity in an open CO2 system

Abstract. The activity of the green alga Scenedesmus obliquus was studied in simplified nutrient solutions (20 mol m₋₃ NaNO₃, 20 mol m₋₃ NH₄C1, 20 mol m₋₃ NH₄NO₃, and 20 mol m₋₃ NaCl, respectively) at 25 °C. The experiments were performed under welldefined incident photon density fluxes ranging from 10 to 200 μmol m₂ s₋₁, Light-dependent changes in pH and alkalinity (A) were followed by means of a potentiometric method using a glass electrode. In the experiments, carbon dioxide with known partial pressure was bubbled through the algal suspension, and during dark periods ul intervals of 1 h, the solution was allowed to equilibrate with the gas phase. This technique was applied to calculate equilibrium values of pH and alkalinity at regular intervals during a 12-h period. Results obtained in NaNO₃, solution show a linear increase in A with time, at each level of illumination studied. After an initial drop, A also increases in NH₄NO₃, solution in a similar way to that in NaNO₃ solution. The change in A with time was also found to increase linearly with the photon density flux studied and no saturation level could be defined. In experiments in NaCl solution, no changes in A were registered while measurements in NH₄Cl solution showed a decrease in A with time.     

Certain Aspects of the Sexuality of Two Species of Chlamydomonas

SUMMARY. When older cultures (18 days old) of Chlamydomonas eugametos were mated, zygote formation occurred under conditions similar to those devised for C. moewusii. Young (6 days old) cultures of the former did not mate when the nitrogen concentration of the medium was high (0.03% NH₄NO₃). In confirmation of the work of Sager and Granick, it was found that low nitrogen concentrations, produced by decreasing the concentration of NH₄NO₃ in the original medium, by increasing the intensity of the illumination, or by using old cultures, enhanced gametogenesis in C. eugametos. It has also been demonstrated that the two species are compatible with one another, even under conditions which are unfavorable for gametogenesis in C. eugametos alone.     

Physiological effects of long-term exposure to low and moderate concentrations of atmospheric NH3 on poplar leaves

Abstract. Poplar shoots ( Populus euramericana L.) obtained from cuttings were exposed for 6 or 8 weeks to NH₃ concentrations of 50 and 100 μgm⁻³ or filtered air in fumigation chambers. After this exposure the rates of NH₃ uptake, transpiration, CO₂ assimilation and respiration of leaves were measured using a leaf chamber. During the long-term exposure also modulated chlorophyll fluorescence measurements were carried out to obtain information about the photosynthetic performance of individual leaves. Both fluorescence and leaf chamber measurements showed a higher photosynthetic activity of leaves exposed to 100 μg NH₃ m⁻³. These leaves showed also a larger leaf conductance and a larger uptake rate of NH₃ than leaves exposed to 50 μg m⁻³ NH₃ or filtered air. The long-term NH₃ exposure did not induce an internal resistance against NH₃ transport in the leaf, nor did it affect the leaf cuticle. So, not only at a short time exposure, but also at a long-term exposure NH₃ uptake into leaves can be calculated from data on the boundary layer and stomatal resistance for H₂O and ambient NH₃-concentration. Furthermore, the NH₃ exposure had no effect on the relation between CO₂-assimilation and stomatal conductance, indicating that NH₃ in concentrations up to 100 μg m⁻³ has no direct effect on stomatal behaviour; for example, by affecting the guard or contiguous cells of the stomata.     

Growth, photosynthesis, and respiration of Chlorella sorokiniana after N-starvation. Interactions between light, CO2 and NH4+ supply

N-sufficient cells of Chlorella sorokiniana Shihira and Krauss, strain 211/8k, absorbed NH₄⁺ under light plus CO₂ conditions, when growth occurred, but not in darkness or in the absence of CO₂, when growth was inhibited. N-sufficient cells subjected to conditions of N-starvation for a 24-h period showed a marked loss of photosynthetic activity. Upon supply of NH₄⁺, N-starved cells sufflated with CO₂ air exhibited a time-dependent recovery of photosynthetic activity, both when suspended in light and in darkness. By contrast, growth only occurred in cells suspended in light. N-starved cells absorbed NH₄⁺ in darkness, but at a lower rate than in light. All of these data suggest that dark NH₄⁺ uptake is driven by N assimilation to recover from N-starvation and that the light-dependent NH₄⁺ uptake is driven by growth, being then influenced by conditions that affect recovery or growth. Unlike CO₂ conditions, in a CO₂-free atmosphere, absorption of NH₄⁺ by N-starved cells occurred at a higher rate in darkness than in light. Accordingly, resumption of photosynthetic potential after NH₄⁺ supply occurred in darkened cells, but not in illuminated cells. Respiratory activity of N-starved cells was enhanced up to 3-fold by NH₄⁺ and 2-fold by methylammonium, with different patterns, suggesting that respiratory enzymes were affected by N-metabolism, especially through short-term control mechanisms triggered by the expenditure of metabolic energy involved in N-metabolism.     

Different effects of supplied ammonium on glutamine synthetase activity in mustard (Sinapis alba) and pine (Pinus sylvestris) seedlings

The activity of glutamine synthetase (GS) in mustard ( Sinapis alba L.) and Scots pine ( Pinus sylvestris L.) seedlings was used as an index to evaluate the capacity to cope with excessive ammonium supply. In these 2 species GS activity was differently affected by the application of nitrogen compounds (NH₄⁺ or NO₃⁻). Mustard seedlings older than 5 days showed a considerable increase in GS activity after NH₄⁺ or NO₃⁻ application. This response was independent of the energy flux, but GS activity in general was positively affected by light. Endogenous NH₄⁺ did not accumulate greatly after nitrogen supply. In contrast, seedlings of Scots pine accumulated NH₄⁺ in cotyledons and roots and showed no stimulation of GS activity after the application of ammonium. In addition, root growth was drastically reduced. Thus, the pine seedlings seem to have insufficient capacity to assimilate exogenously supplied ammonium. NO₃⁻, however, did not lead to any harmful effects.     

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.     

Effect of different nitrogen nutrients on the viability, protein synthesis and tannin production of Scots pine callus

The effect of two different media on the growth, metabolism and viability of Scots pine ( Pinus sylvestris ) callus cultures was studied. Inorganic nitrogen in the culture media (modified MS) was in the form of either KNO₃ or NH₄NO₃. The cultures were started from buds of mature Scots pine. Growth was poor on the medium with KNO₃, but this compound had a noticeable effect on the metabolism of the callus, which was reflected in alterations in protein and polyphenol synthesis and the pH of the culture medium. Although the fresh mass, water content and viability of the callus decreased when KNO₃ was the exclusive inorganic nitrogen nutrient, protein synthesis was more abundant. Electrophoretic analyses indicated alterations in the patterns of soluble proteins and purified glycoproteins. Phenylalanine ammonia-lyase (EC 4.3.1.5) activities were high in all the calluses, and concentrations of condensed tannins and their precursors, catechins, were higher than in intact buds. The role of inorganic nitrogen nutrition in the deterioration of tissues is discussed on the basis of the effect of ammonium on the metabolism of pine callus.     

Impact of gaseous nitrogen deposition on plant functioning

Dry deposition of NH₃ and NO_x (NO and NO₂) can affect plant metabolism at the cellular and whole-plant level. Gaseous pollutants enter the plant mainly through the stomata, and once in the apoplast NH₃ dissolves to form NH₄⁺, whereas NO₂ dissolves to form NO₃⁻ and NO₂⁻. The latter compound can also be formed after exposure to NO. There is evidence that NH₃-N and NO_x-N can be reversibly stored in the apoplast. Temporary storage might affect processes such as absorption rate, assimilation and re-emission. Once formed, NO₃⁻ and NO₂⁻ can be reduced, and NH₄⁺ can be assimilated via the normal enzymatic pathways, nitrate reductase (NR), nitrite reductase and the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. Fumigation with low concentrations of atmospheric NH₃ increases in vitro glutamine synthetase activity, but whether this involves both or only one of the GS isoforms is still an open question. There seems to be no correlation between fumigation with low concentrations of NH₃ and in vitro GDH activity. The contribution of atmospheric NH₃ and NO₂ deposition to the N budget of the whole plant has been calculated for various atmospheric pollutant concentrations and relative growth rates ( RGRs ). It is concluded that at current ambient atmospheric N concentrations the direct impact of gaseous N uptake by foliage on plant growth is generally small.