A new class of arginine-requiring mutants in Chlamydomonas reinhardi

Summary Forward mutations to auxotrophy have been induced in Chlamydomonas reinhardi after treatment of wild-type cells with ethyl methanesulfonate (EMS) and plating on a selective medium supplemented with yeast extract but deprived of ammonium chloride.Among the 41 stable mutants isolated, 6 were arginine-requiring. Reconstruction experiments show that the growth of all these arginine auxotrophs is inhibited on media containing high concentrations of NH ₄ ⁺ ions. It is concluded that the isolation of this new class of mutants was made possible because of the low ammonium concentration of the plating medium.It appears therefore that in Chlamydomonas, as in other microorganisms, the specificity of mutations may be imposed by the selective medium.Chargé de Recherches du Fonds National de la Recherche Scientifique.     

Arginine metabolism in Chlamydomonas reinhardi. Conditional expression of arginine-requiring mutants

Summary In Chlamydomonas reinhardi, the arginine-requiring mutants directly derived from the wild-type strain are not able to grow on media containing the routinely used concentration of ammonia. The biochemical basis of this phenomenon has been investigated.The results show that the uptake of arginine is severely depressed by NH ₊ ⁴ and that arginine which succeeds in entering the cells under these conditions is immediately broken down by the first enzyme of the catabolic pathway, arginine deiminase.Accordingly, growth is inhibited as the result of non-availability of exogenous arginine for protein synthesis.It is suggested that similar interactions could be responsible for the lack of other types of amino acid-requiring mutants in Chlamydomonas.Chargé de Recherches du Fonds National Belge de la Recherche Scientifique.     

Intracellular measurement of ammonium in Chara corallina using ion-selective microelectrodes

The study of ammonium (NH₄ ⁺) transport across plant cell membranes requires accurate measurement of NH₄ ⁺ gradients across subcellular gradients. We have developed an ammonium-selective microelectrode based on the ionophore nonactin. This electrode can detect NH₄ ⁺ activities (a_NH4) in vivo in the millimolar range in the presence of cytosolic levels of potassium, the main interfering ion. The electrode was used to measure intracellular a_NH4 in internodal cells of the giant alga Chara corallina. Results from cells incubated in media supplemented with 1 mM NH₄ ⁺ produced two populations, with means of 7.3 and 30.8 mM, respectively. HPLC analysis of vacuolar sap suggests the higher population represents vacuolar impalements, and the lower population can thus be assumed to be cytosolic. These results suggest a four-fold accumulation of NH₄ ⁺ in the vacuolar compartment of Chara. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nitrogen regulation of gibberellin biosynthesis in Gibberella fujikuroi

Summary In Gibberella fujikuroi, ammonium (NH₄ ⁺) interfered with the production of gibberellic acid (GA₃). Optimal production occurred at 19 mm (NH₄)₂SO₄ and the synthesis of GA₃ was reduced threefold in a medium with 38 mm (NH₄)₂SO₄. Using a resting cell system with mycelia previously grown on two concentrations (19 mm and 38 mm) of (NH₄)₂SO₄, it was found that NH₄ ⁺ depressed synthesis of the gibberellin-synthesizing enzymes. Furthermore, addition of NH₄ ⁺ to a producing system shut off gibberellin formation, indicating that the negative effect of NH₄ ⁺ ions is also due to inhibition of one or more enzymes in the gibberellin biosynthesis pathway. The onset of gibberellin biosynthesis in media with high (38 mm) and low (19 mm) concentrations of (NH₄)₂SO₄ was studied by addition of cycloheximide to batch cultures of various ages. Offprint requests to: B. Brückner     

Effect of ammonium ions and cytokinins on hyperhydricity and multiplication rate of in vitro regenerated shoots of <Emphasis Type="Italic">Aloe polyphylla</Emphasis>

In search for the optimal culture conditions resulting in a high production of healthy plants and low occurrence of hyperhydricity in tissue cultured regenerants of Aloe polyphylla, we investigated the relationship between ammonium ions in the medium, applied cytokinins (CKs) and CK concentrations in the induction of hyperhydricity. Shoots were grown on media with different NH₄ ⁺ concentrations (10.3, 20.6 and 61.8 mM) and supplemented with N⁶-benzyladenine (BA), zeatin or thidiazuron (TDZ) at 0, 5 or 15 μM. Elevating the levels of NH₄ ⁺, in the absence of CKs, could not induce hyperhydricity. Similarly, very low hyperhydricity was observed when CKs were added to media containing low NH₄ ⁺ (10.3 mM). However, in the presence of higher NH₄ ⁺ concentrations, CKs increased hyperhydricity in a concentration-dependant manner, suggesting that they were capable of inducing this syndrome only when other factors in the culture system were not optimised. High numbers of healthy looking shoots were produced on media with low NH₄ ⁺ and low BA or zeatin (5 μM). The use of TDZ resulted in the formation of buds, which did not develop into shoots. Identifying the factors responsible for hyperhydricity is an important step in the successful use of the micropropagation technique for the conservation of this species.     

Regulation of NH4 + transport pathways in Pisum arvense roots

The effects of metabolic and protein synthesis inhibitors on NH₄ ⁺ uptake by Pisum arvense plants at low (0.05 mM) and high (1 mM) external ammonium concentration were studied. In short-time experiments cycloheximide decreased the ammonium uptake rate at low level of NH₄ ⁺ and increased the absorption of NH₄ ⁺ from uptake medium containing high ammonium concentration. Arsenate and azide supplied into uptake solutions at low ammonium concentration strongly decreased or completely suppressed the NH₄ ⁺ uptake rate, respectively. When the experiments were carried out at high level of ammonium only azide decreased the uptake rate of NH₄ ⁺ and arsenate stimulated this process. Dinitrophenol very strongly repressed the uptake rate of NH₄ ⁺ at both ammonium concentrations. After removing dinitrophenol from both solutions, neither at low nor high external ammonium level the recovery of NH₄ ⁺ uptake rate was achieved within 150 min or 3 h, respectively. The recovery of NH₄ ⁺ uptake rate after removing azide was observed within 90 min and 3 h at low and high ammonium concentrations, respectively. The regulation of NH₄ ⁺ uptake by some inhibitors at low external ammonium level was investigated using plasma membrane vesicles isolated from roots by two-phase partitioning. Orthovanadate completely suppressed the uptake of NH₄ ⁺ by vesicles and quinacrine decreased the NH₄ ⁺ uptake which 55 suggests that ammonium uptake depends on activities of plasma membrane-bound enzymes. On the other hand, it was found that dinitrophenol completely reduced the NH₄ ⁺ uptake by vesicles. The various effects of inhibitors on ammonium uptake dependent on external ammonium concentration suggest the action of different ammonium transport systems in Pisum arvense roots. The ammonium transport into root cells at low NH₄ ⁺ level requires energy and synthesis of protein in the cytoplasm. The research was supported by grant of KBN No. 6PO4C 068 08     

The effect of total inorganic nitrogen and the balance between its ionic forms on adventitious bud formation and callus growth of ‘Węgierka Zwykła’ plum (<Emphasis Type="Italic">Prunus domestica</Emphasis> L.)

The effect of different ammonium NH ₄ ⁺ and nitrate NO ₃ ^? ratios (4:1, 2:1, 1:1, 1:2, 1:4, 1:6) on organogenesis of ‘W?gierka Zwyk?a’ leaf explants cultivated on media with nitrogen levels equalling full- or half-MS was investigated. On media with total nitrogen equal to ½ MS, explant regeneration increased significantly and was highest on media with 1:2 or 1:4 NH ₄ ⁺ :NO ₃ ^? ratio. An excess of ammonium versus nitrate ions had a negative effect on both regeneration and biomass. Addition of potassium to the medium increased the fresh weight of explants and the number of adventitious buds.     

γ‐Aminobutyric acid addition alleviates ammonium toxicity by limiting ammonium accumulation in rice (Oryza sativa) seedlings

Excessive use of nitrogen (N) fertilizer has increased ammonium (NH₄⁺) accumulation in many paddy soils to levels that reduce rice vegetative biomass and yield. Based on studies of NH₄⁺ toxicity in rice (Oryza sativa, Nanjing 44) seedlings cultured in agar medium, we found that NH₄⁺ concentrations above 0.75 mM inhibited the growth of rice and caused NH₄⁺ accumulation in both shoots and roots. Use of excessive NH₄⁺ also induced rhizosphere acidification and inhibited the absorption of K, Ca, Mg, Fe and Zn in rice seedlings. Under excessive NH₄⁺ conditions, exogenous γ‐aminobutyric acid (GABA) treatment limited NH₄⁺ accumulation in rice seedlings, reduced NH₄⁺ toxicity symptoms and promoted plant growth. GABA addition also reduced rhizosphere acidification and alleviated the inhibition of Ca, Mg, Fe and Zn absorption caused by excessive NH₄⁺. Furthermore, we found that the activity of glutamine synthetase/NADH‐glutamate synthase (GS; EC 6.3.1.2/NADH‐GOGAT; EC1.4.1.14) in root increased gradually as the NH₄⁺ concentration increased. However, when the concentration of NH₄⁺ is more than 3 mM, GABA treatment inhibited NH₄⁺‐induced increases in GS/NADH‐GOGAT activity. The inhibition of ammonium assimilation may restore the elongation of seminal rice roots repressed by high NH₄⁺. These results suggest that mitigation of ammonium accumulation and assimilation is essential for GABA‐dependent alleviation of ammonium toxicity in rice seedlings.     

Caesium uptake and toxicity in the cyanobacterium <Emphasis Type="Italic">Nostoc muscorum</Emphasis>

A NH₄⁺ transport-defective mutant and a K⁺ transport-defective mutant of the cyanobacterium Nostoc muscorum were analysed with regard to percentage survival as a function of CsCl toxicity and Cs⁺ uptake activity. Neither survival nor Cs⁺ uptake was affected in either of the two mutants when compared with the wild type. The results indicate that the toxicity of Cs⁺ is determined at more than one cellular site in this organism.     

Ammonium-Stimulated Root Hair Branching is Enhanced by Methyl Jasmonate and Suppressed by Ethylene in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Root hair development is orchestrated by nutritional factors and plant hormones. We investigated the action of ammonium (NH₄⁺) and its interactions with methyl jasmonate (MeJA) and ethylene in Arabidopsis root hair growth. The formation of root hair branches was dramatically stimulated in media containing 1.25 to 20 mM NH₄⁺ at pH values of 4.0 to 6.5. The NH₄⁺-treated root hairs showed a very short tip growth stage and swells on the sides that indicated the emergence of branches. MeJA (0.08 to 10 μM) worked in synergism with NH₄⁺ to enhance hair branching. In contrast, ethylene had an antagonistic effect; the stimulation of hair branching by NH₄⁺ was suppressed by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and was diminished in ethylene-overproducing mutant eto1-1 seedlings. Moreover, the application of Ag⁺, an ethylene inhibitor, reduced the ACC-induced inhibition of NH₄⁺-stimulated hair branching and restored NH₄⁺-stimulated hair branching in eto1-1 seedlings. Thus, the actions of jasmonate and ethylene appear to be dependent on nutritional conditions such as available nitrogen.     

Regulation of the high-affinity ammonium transporter (BnAMT1;2) in the leaves of Brassica napus by nitrogen status

Substantial concentrations of NH₄ ⁺ are found in the apoplast of the leaves of Brassica napus. Physiological studies on isolated mesophyll protoplasts with ¹⁵NH₄ ⁺ revealed the presence of a high-affinity ammonium transporter that shared physiological similarity to the high-affinity NH₄ ⁺ transporters in Arabidopsis thaliana (AtAMT1;3). PCR techniques were used to isolate a full-length clone of a B. napus homologue of AMT1 from shoot mRNA which showed 97% similarity to AtAMT1;3. The full-length cDNA when cloned into the yeast expression vector pFL61 was able to complement a yeast mutant unable to grow on media with NH₄ ⁺ as the sole nitrogen source. Regulatory studies with detached leaves revealed a stimulation of both NH₄ ⁺ uptake and expression of mRNA when the leaves were supplied with increasing concentrations of NH₄ ⁺. Withdrawal of NH₄ ⁺ supply for up to 96 h had little effect on mRNA expression or NH₄ ⁺ uptake; however, plants grown continuously at high NH₄ ⁺ levels exhibited decreased mRNA expression. BnAMT1;2mRNA expression was highest when NH₄ ⁺ was supplied directly to the leaf and lowest when either glutamine or glutamate was supplied to the leaves, which directly paralleled chloroplastic glutamine synthetase (GS2) activity in the same leaves. These results provide tentative evidence that BnAMT1;2may be regulated by similar mechanisms to GS2 in leaves.     

In situ removal of ammonium ions from hybridoma cell culture media: Selection of adsorbent

Various ion-exchange resins and zeolite Phillipsite-Gismondine were tested for their potential application as adsorbents for thein situ removal of ammonium ions from animal cell culture media in attempting to increase hybridoma cell density and MAb productivity. A zeolite, Phillipsite-Gismondine demonstrated the best performance in terms of NH₄ ⁺ adsorption capacity, selectivity and autoclavability among the various adsorbents tested. The biocompatibility of Phillipsite-Gismondine was also tested and the result showed improvement in cell density, viability, and antibody productivity.     

Ammonium and Methylammonium Transport in Egeria densa Leaves in Conditions of Different H+ Pump Activity

Previous data in Egeria densa leaves demonstrated a strong inhibitory effect of Cs⁺ on passive K⁺ influx and on K⁺-induced, ATP-dependent electrogenic proton extrusion. In this paper we analyzed, using the same material, the effects of Cs⁺ on ammonium (NH₄⁺) and methylammonium (CH₃NH₃⁺) transport in order to elucidate whether a common transport system for K⁺ and NH₄⁺ could be demonstrated. The effects of Cs⁺ on NH₄⁺- and CH₃NH₃⁺-induced titratable H⁺ extrusion (–ΔH⁺) and on transmembrane electrical potential difference (E_m) in E. densa leaves were analyzed in parallel. All experiments were run either in the absence or presence of fusicoccin, corresponding to low or high H⁺-ATPase activity and membrane hyperpolarization and leading, in this material, to respectively active or passive transport of K⁺. The results suggest the presence in E. densa leaves of two distinct pathways for NH₄⁺ uptake: one in common with NH₄⁺ and (with lower affinity) CH₃NH₃⁺, insensitive to Cs⁺, and a second system, operating at higher H⁺-ATPase activity and E_m hyperpolarization, strongly inhibited by Cs⁺ and impermeable to CH₃NH₃⁺. In agreement with this hypothesis, Xenopus laevis oocytes injected with the KAT1 RNA of Arabidopsis thaliana were permeable to K⁺ and NH₄⁺, but not to CH₃NH₃⁺.     

MSX-resistant mutants of Anabaena 7120 with derepressed heterocyst development and nitrogen fixation

To investigate the role of ammonium-assimilating enzyme in heterocyst differentiation, pattern formation and nitrogen fixation, MSX-resistant and GS-impaired mutants of Anabaena 7120 were isolated using transposon (Tn5-1063) mutagenesis. Mutant Gs1 and Gs2 (impaired in GS activity) exhibited a similar rate of nitrogenase activity compared to that of the wild type under dinitrogen aerobic conditions in the presence and absence of MSX. Filaments of Gs1 and Gs2 produced heterocysts with an evenly spaced pattern in N₂-grown conditions, while addition of MSX altered the interheterocyst spacing pattern in wild type as well as in mutant strains. The wild type showed complete repression of heterocyst development and nitrogen fixation in the presence of NO₃ ^– or NH₄ ⁺, whereas the mutants Gs1 and Gs2 formed heterocysts and fixed nitrogen in the presence of NO₃ ^– and NH₄ ⁺. Addition of MSX caused complete inhibition of glutamine synthetase activity in wild type but Gs1 and Gs2 remained unaffected. These results suggest that glutamine but not ammonium is directly involved in regulation of heterocyst differentiation, interheterocyst spacing pattern and nitrogen fixation in Anabaena.     

The effect of ammonium on assimilatory nitrate reduction in the haloarchaeon Haloferax mediterranei

Physiology, regulation and biochemical aspects of the nitrogen assimilation are well known in Prokarya or Eukarya but they are poorly described in Archaea domain. The haloarchaeon Haloferax mediterranei can use different nitrogen inorganic sources (NO₃⁻, NO₂⁻ or NH₄⁺) for growth. Different approaches were considered to study the effect of NH₄⁺ on nitrogen assimilation in Hfx. mediterranei cells grown in KNO₃ medium. The NH₄⁺ addition to KNO₃ medium caused a decrease of assimilatory nitrate (Nas) and nitrite reductases (NiR) activities. Similar effects were observed when nitrate-growing cells were transferred to NH₄⁺ media. Both activities increased when NH₄⁺ was removed from culture, showing that the negative effect of NH₄⁺ on this pathway is reversible. These results suggest that ammonium causes the inhibition of the assimilatory nitrate pathway, while nitrate exerts a positive effect. This pattern has been confirmed by RT-PCR. In the presence of both NO₃⁻ and NH₄⁺, NH₄⁺ was preferentially consumed, but NO₃⁻ uptake was not completely inhibited by NH₄⁺ at prolonged time scale. The addition of MSX to NH₄⁺ or NO₃⁻ cultures results in an increase of Nas and NiR activities, suggesting that NH₄⁺ assimilation, rather than NH₄⁺ per se, has a negative effect on assimilatory nitrate reduction in Hfx. mediterranei. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The effect of some ammonium salts on nitrate reductase level,onin vivo nitrate reduction and on nitrate content in excisedpisum sativum roots

The effect of some ammonium salts on nitrate reductase (NR) level, onin vivo nitrate reduction and on nitrate content was followed in the presence of nitrate in the medium, under changing experimental conditions, in excisedPisum sativum roots, and their effect was compared with that of KNO₃, Ca(NO₃)₂ and NaNO₃ at 15 mM NO₃ ^- concentration, i.e. at a concentration which considerably exceeded the level of saturation with nitrate with respect to nitrate reductase. The effect of ammonium salts on NR level is indirect and changes from a positive one to a strongly negative one which is dependent on the time of action of the salt, on the presence of other cations, on pH of the solution of the ammonium salt and on the nature of the anion of the ammonium salt. A positive effect on the enzyme level can be observed in the presence of other cations than NH₄ ⁺ at suitable concentrations of those ammonium salts, the solutions of which have their pH values in the acid region (i.e. NH₄H₂PO₄, (NH₄)₂SO₄ and NH₄NO₃). However their positive effect is independent of the presence of NH₄ ⁺ ions, and it is obviously the result of an increased concentration of H⁺ ions. A clear-cut negative effect on NR level can be observed after 24 h in one-salt NH₄NO₃ solution where NH₄ ⁺ is not balanced with other cations and thus certainly can adversely influence many metabolic processes, and in the solutions containing neutral (pH 6.2) and dibasic ammonium phosphates in which dissolved undissociated ammonia [(NH₃). (H₂O) which can also affect many metabolic processes incl. proteosynthesis] probably has a toxic influence. Thein vivo nitrate reduction is always depressed in excised pea roots in the presence of ammonium salts in the medium, regardless of the level of nitrate reductase. Under the described conditions, no relationship could be established between the enzyme level and the so-called metabolic NO₃ ^- pool (i.e. NO₂ ^- production under anaerobic conditions), nor between NR level and the total nitrate content in the roots. One-salt solutions of NaNO₃, Ca(NO₃)₂ and KNO₃ exert different effects on the level of nitrate reductase and on the content of NO₃ ^- in the roots, but the in vivo NO₃ ^- reduction shows the same trend as NR level in the roots influenced by these salts. Cl^- ions, supplied in NH₄C1, depress both NR level and NO₃ ^- content in the roots at higher concentrations, but they do not significantly affect the in vivo nitrate reduction in comparison with other ammonium salts. These results indicate that NR level,in vivo nitrate reduction, and nitrate uptake can be regulated in pea roots independently of each other.     

Altered cell wall properties are responsible for ammonium‐reduced aluminium accumulation in rice roots

The phytotoxicity of aluminium (Al) ions can be alleviated by ammonium (NH₄⁺) in rice and this effect has been attributed to the decreased Al accumulation in the roots. Here, the effects of different nitrogen forms on cell wall properties were compared in two rice cultivars differing in Al tolerance. An in vitro Al‐binding assay revealed that neither NH₄⁺ nor NO₃^? altered the Al‐binding capacity of cell walls, which were extracted from plants not previously exposed to N sources. However, cell walls extracted from NH₄⁺‐supplied roots displayed lower Al‐binding capacity than those from NO₃^?‐supplied roots when grown in non‐buffered solutions. Fourier‐transform infrared microspectroscopy analysis revealed that, compared with NO₃^?‐supplied roots, NH₄⁺‐supplied roots possessed fewer Al‐binding groups (‐OH and COO‐) and lower contents of pectin and hemicellulose. However, when grown in pH‐buffered solutions, these differences in the cell wall properties were not observed. Further analysis showed that the Al‐binding capacity and properties of cell walls were also altered by pHs alone. Taken together, our results indicate that the NH₄⁺‐reduced Al accumulation was attributed to the altered cell wall properties triggered by pH decrease due to NH₄⁺ uptake rather than direct competition for the cell wall binding sites between Al³⁺ and NH₄⁺.     

Some properties of a Klebsiella pneumoniae ammonium transport negative mutant (Amt−)

The main property of an Amt^- (ammonium transport negative) mutant of Klebsiella pneumoniae is its inability to accumulate NH ₄ ⁺ intracellularly. When growing on nitrogen sources other than NH ₄ ⁺ , the mutant constantly looses NH₃ by diffusion. This loss results in poor growth. The NH₃ excretion suggests the existence of a futile cycle (NH₃ loss/NH ₄ ⁺ reabsorption) in the wild type and possibly other bacterial strains, which do not constantly excrete NH₃.Dedicated to Prof. R. H. Burris     

Molecular Dynamics Simulation Studies of Zeolite-A. IV. Structure and Dynamics of NH4 + ions in a Rigid Dehydrated Zeolite-A

Abstract

In recent papers [1–3] we reported molecular dynamics simulation studies of ions and water molecules adsorbed in a rigid zeolite-A framework using a simple Lennard-Jones potential plus Coulomb potential with Ewald summation to investigate the structure and dynamics of the adsorbates. In the present paper the same technique is applied to study the local structure and dynamics of NH₄ ⁺ ions in a rigid dehydrated zeolite-A. During the preliminary equilibration, the unstable NH₄(4) type ion (the 12th ion) is pushed down to near a more stable 6-ring position in the α-cage that is already associated with an NH₄(1) type ion (the 1st) in the β-cage, which moves to another 6-ring position in the β-cage that is already associated with an NH₄(2) type ion (the 7th) in the α-cage. Calculated x, y, and z coordinates of some NH₄ ⁺ ions are in good agreement with those obtained from an X-ray diffraction experiment except that no NH₄(4) type ion is found and there are six NH₄(2) type ions instead of 0.5 and 5.5 occupancy. The analyses of calculated interatomic distances and time correlation functions of these ions indicate that the NH₄(1 – 1) and NH₄(3) type ions are associated loosely with only one O (3) atom of the 6-ring and with only one O (1) atom of the 8-ring windows, respectively, while the NH₄(1–2) and NH₄(2) type ions are associated strongly with two or three O (3) atoms of the 6-ring windows in the α- and β-cages, respectively. The analysis of hydrogen bond time correlation functions of these ions indicate that about one, two or three, three, and one hydrogen bond of each NH₄(1–1), NH₄(1–2), NH₄(2) and NH₄(3) type ion is kept for 1.4, 21, 75, and 1.4 ps, respectively, before breakup of the hydrogen bond occurs and significant exchange of O atom hydrogen-bonded to the ion.