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XsiAMT1.1a was identified as a novel ammonium uptake functional gene and its overexpression combined with GA4 application significantly increased yield in Arabidopsis thaliana.

Nitrogen (N) is a key limiting factor for plant yield. Ammonium is one of the main N forms absorbed by plants. Overexpression of ammonium uptake functional genes, such as ammonium transporter (AMT), can increase yield. However, the AMTs reported to enhance yield significantly is still limited. No researches have focused on the effect of overexpressing AMT combined with hormone application on yield improvement. In this study, we first investigated the role of XsiAMT1.1a, a potential ammonium uptake functional gene in an ammonium preference plant Xanthium sibiricum, in ammonium uptake by the analysis of bioinformatics, gene expression and subcellular localization, and the determination of ammonium uptake rate in endogenous silencing and heterologous overexpression plants. Subsequently, the effect of XsiAMT1.1a overexpression combined with hormone application on yield increase was further investigated in model plant Arabidopsis thaliana. Our results showed that XsiAMT1.1a shared the same conserved domains with AtAMT1 subfamily members and localized on the plasma membrane. XsiAMT1.1a was induced by N deficiency and highly expressed during the reproductive period. XsiAMT1.1a endogenous silencing and heterologous overexpression significantly decreased and increased ammonium uptake rates in X. sibiricum and A. thaliana, respectively. Overexpression of XsiAMT1.1a significantly improved total N accumulation, biomass and yield in A. thaliana, while XsiAMT1.1a overexpression combined with GA4 application had a stronger promoting effect on the above indicators. Our research identified a novel ammonium uptake functional gene, XsiAMT1.1a, and provided a new yield-increasing strategy which was verified in A. thaliana.

Arabidopsis

Ammonium uptake by nitrogen fixing bacteria I. Azotobacter vinelandii.

Both the changes in the activities of nitrogenase, glutamine synthetase and glutamate dehydrogenase and in the extracellular and intracellular NH4+ concentrations were investigated during the transition from an NH4+ free medium to one containing NH4+ ions for a continuous culture of Azotobacter vinelandii. If added in amounts causing 80-100% repression of nitrogenase, ammonium acetate, lactate and phosphate are absorbed completely, whereas chloride, sulfate and citrate are only taken up to about 80%. After about 1-2 hrs the NH4+ remaining in the medium is absorbed too, indicating the induction or activation of a new NH4+ transport system. One of the new permeases allows the uptake of citrate in the presence of sucrose. Addition of inorganic NH4+ level leads to a reversible rise in the glutamine synthetase activity which is not prevented by chloramphenicol, and to a reversible decrease in nitrogenase activity. During these measurements glutamate dehydrogenase activity remains close to zero. The intracellular NH4+ level of about 0.6 mM does not change when extracellular NH4+ is taken up and repression of nitrogenase starts.

Acetates

Ammonium uptake and metabolism by mitrogen fixing bacteria. II. Klebsiella pneumoniae.

The primary steps of N2, ammonia and nitrate metabolism in Klebsiella pneumoniae grown in a continuous culture are regulated by the kind and supply of the nitrogenous compound. Cultures growing on N2 as the only nitrogen source have high activities of nitrogenase, unadenylated glutamine synthetase and glutamate synthase and low levels of glutamate dehydrogenase. If small amounts of ammonium salts are added continuously, initially only part of it is absorbed by the organisms. After 2-3 h complete absorption of ammonia against an ammonium gradient coinciding with an increased growth rate of the bacteria is observed. The change in the extracellular ammonium level is paralleled by the intracellular glutamine concentration which in turn regulates the glutamine synthesis and an induction of glutamate dehydrogenase synthesis. Upon deadenylation these events are reversed.--Addition of dinitrophenol causes transient leakage of intracellular ammonium into the medium.

Biological Transport

Depression of uracil uptake by ammonium in Neurospora crassa.

The mechanism of uracil uptake and one aspect of its regulation were studied in germinated conidia of Neurospora crassa. Uracil was found to be taken up by a transport mechanism that did not exhibit Michaelis-Menten kinetics. Rather, the kinetic patterns indicated two separate systems or a single transport mechanism with negative cooperativity. Cytosine and thymine inhibited uracil uptake, but uridine did not. The mutant strain uc-5-pyr-1, which failed to transport uracil, was used in reversion studies and to map the uc-5 locus. Spontaneous reversion rates at the uc-5 locus were found to be approximately 2 x 10(-8), indicating that the uc-5 lesion results from a single mutation. Loss of the uracil transport function through a single mutation favors the model of a single transport mechanism with negative cooperativity. Uracil uptake was significantly decreased in the presence of NH 4+, and evidence is presented for repression by NH4+ of a uracil transport system. Growth rates of pyrimidine-requiring and wild-type strains measured in the presence and absence of NH4+, with uracil as the pyrimidine supplement, showed that NH4+ decreased the growth rates of the pyrimidine-requiring strains significantly, while having no effect on wild-type growth rates.

Ammonia

The effect of pH and amino acids on conidiation and pigment production of Monascus major ATCC 16362 and Monascus rubiginosus ATCC 16367 in submerged shaken culture.

Monascus major ATCC 16362 and Monascus rubiginosus ATCC 16367 were cultivated aerobically on media containing nitrate or ammonium as nitrogen source to which the following modifications were made: (1) pH adjusted to 2.5 before sterilization; (2) addition of yeast extract; (3) addition of amino acids in identical proportions and concentrations to those found in yeast extract; (4) adjustment of pH to 2.5 after addition of amino acids. The addition of amino acids in the form of yeast extract increased mycelium formation and reduced conidiation and pigment production. The addition of an amino acid mixture did not increase mycelium formation to the same extent as yeast extract but increased the number of conidia, while pigment production was reduced, especially when nitrate was the nitrogen source. As the amino acids are taken up after conidial formation has started, it would appear that it is not the amino acids themselves which are directly responsible for the induction of conidiation. The addition of amino acids inhibits nitrate and ammonium uptake suggesting the need for an early intracellular nitrogen limitation to induce conidiation. Lowering the pH inhibits the formation of conidia and increases pigment production; also the effect of amino acid addition is totally annulled. The pH of the medium is all important in regulating the formation of conidia and pigment production. The possible effects of the pH on the uptake of certain medium components is discussed, as well as their possible control of certain metabolic pathways which ultimately determines the availability of intermediates for conidiation and pigment production.

Aerobiosis

Comparative uptake of fluoride from sodium fluoride, ammonium fluoride, and barium fluoride in rat teeth when predominantly administered in the pre-eruptive stage of development.

A comparative study was undertaken which focused on the systemic uptake of sodium fluoride (NaF), ammonium fluoride (NH4F), and barium fluoride (BaF2) in rat pups. Two critical achievements made this comparative study possible: (a) the demonstration of significant increases in fluoride (F) uptake-deposition in the treatment group pups relative to control group rats as a result of stomach tube feeding; and (b) the demonstration of clear-cut differences in F concentration levels between the treatment groups as a result of this stomach tube-systemic uptake. Data were reliable and significant enough to suggest that, of the 3 compounds in question, NH4F is absorbed most successfully in a systemic fashion, whereas BaF2 is the least absorbed.

Ammonia

Assimilation of ammonia and growth of biotin deficient Aspergillus nidulans.

Biotin deficiency in Aspergillus nidulans has been found to increase the uptake of ammonium ions, associated with a marked increase in the activity of NADP-linked glutamate dehydrogenase, which is found to be the major route of ammonia assimilation in this culture. The results obtained are discussed with respect to the growth of Aspergillus nidulans during biotin deficiency.

Alanine

Regulation of ammonia uptake in aspergillus nidulans.

The ammonia uptake in A. nidulans was found to be linear for about 20 min, and was proportional up to 1.5 mg/ml dry cell density. The transport of ammonia does not involve energy. Normal and biotin deficient A. nidulans showed an identical Km-values of 10.26 X 10(-5) M ammonia for uptake. The uptake of ammonium ion has been shown to be regulated by the intracellular concentration of ammonia.

Aspergillus nidulans

Uptake of [14C]methylammonium by plankton communities: a comparative assay for ammonium transport systems in natural waters.

A diverse range of freshwater plankton communities were tested for their ability to take up [14C]methylammonium. Uptake occurred at low substrate levels by high-affinity, energy-requiring, transport systems which were competitively inhibited by ammonium but not by L-amino acids or nicotinamide. A simple competitive inhibition model was used to examine the effects of increasing ammonium levels on uptake in a eutrophic lake. Apparent K1 values for the labelled substrate markedly increased with increasing ammonium. The transport systems had an approximately five-fold greater affinity for ammonium than for methylammonium. The Vmax for methylammonium uptake was relatively insensitive to large changes in ambient ammonium levels. This kinetic parameter may be a useful comparative measure of ammonium transport capacity in natural waters, particularly where low ambient ammonium concentrations preclude the use of 15N.

Amino Acids

Uptake and degradation of asialo-fetuin by isolated rat hepatocytes.

125I-labelled asialo-fetuin was taken up by isolated rat hepatocytes by a saturable process. Half maximum uptake was seen at about 3 . 10(-8) M asialo-fetuin. Rate of uptake of asialo-fetuin exceeded rate of degradation at all concentrations of asialo-fetuin tested. Degradation of asialo-fetuin, as indicated by release of acid-soluble radioactivity from the cells, was inhibited by NH4Cl and chloroquine. The intracellular distribution of labelled asialo-fetuin was studied by differential and density gradient centrifuging. The distribution curves for radioactivity indicated that asialo-fetuin was present in lysosomes about 1 h after the uptake had started. Chloroquine and ammonium ions seemed to inhibit the uptake of asialo-fetuin into the lysosomes, possibly by interfering with the fusion between phagosomes and lysosomes.

Acetylglucosaminidase

The effect of adrenocorticotropin and nucleotides on Ca2+ uptake in adrenal cortical microsomal vesicles.

Rat and bovine adrenal cortical microsomal fractions isolated at 27,000 x g and 105,000 x g accumulated Ca2+ by a nonmitochondrial, ATP-dependent uptake system that was stimulated by ammonium oxalate. ACTH (2 mU/ml) significantly increased Ca2+ uptake in bovine adrenal cortical microsomes and in adrenal microsomes from acutely hypophysectomized rats, but only when the hormone was preincubated with intact tissue and not when it was added after homogenization. ACTH did not stimulate C2+ uptake in adrenal microsomes isolated from nonhypophysectomized, ether-stressed rats, in which basal Ca2+ uptake was higher than that observed in microsomes from hypophysectomized animals. The peptides oxytocin, insulin, and TSH did not stimulate Ca2+ uptake by adrenal cortical microsomes. ACTH preincubated with intact tissue had no effect on Ca2+ uptake in microsomes from liver, kidney, esophagus, or aorta. cAMP, 5'-AMP, and several other nucleotides, nucleosides, and related compounds stimulated adrenal cortical microsomal Ca2+ uptake by as much as 540% of control. The stimulatory effects of nucleotides, unlike those of ACTH, were apparent even when the agents were added after homogenization. However, like ACTH, the nucleotides were unable to stimulate Ca2+ uptake when they were added to isolated membrane vesicles during Ca2+ uptake measurements. It is suggested that the microsomal Ca2+ uptake system may respond to physiological stimulants and regulate Ca2+ availability in the intact cell.

Adrenal Cortex

Imaging the adrenal medulla with an I-131-labeled antiadrenergic agent.

Tissue distributions of four antiadrenergic agents labeled with iodine-125 have been determined in dogs. [125I] ortho-iodobenzldimethyl-2-hydroxyethyl ammonium and [125I] ortho-iodobenzyldimethylethyl ammonium show highly selective uptake in the adrenal medulla. Studies of molecular structure-distribution indicate that both the nature of the cationic head and the ring position of the iodine atom greatly influence adrenal specificity. Distinct images of dogs' adrenal medulla have been obtained 4 days after i.v. injection of 1.5 mCi of [131I] ortho-iodobenzyldimethyl-2-hydroxyethyl ammonium.

Adrenal Medulla

Inhibition of amino acid transport by ammonium ion in Saccharomyces cerevisiae.

The rate of transport of L-amino acids by Saccharomyces cerevisiae epsilon 1278b increased with time in response to nitrogen starvation. This increase could be prevented by the addition of ammonium sulfate or cycloheximide. A slow time-dependent loss of transport activity was observed when ammonium sulfate (or ammonium sulfate plus cycloheximide) was added to cells after 3 h of nitrogen starvation. This loss of activity was not observed in the presence of cycloheximide alone. In a mutant yeast strain which lacks the nicotinamide adenine dinucleotide phosphate-dependent (anabolic) glutamate dehydrogenase, no significant decrease in amino acid transport was observed when ammonium sulfate was added to nitrogen-starved cells. A double mutant, which lacks the nicotinamide adenine dinucleotide phosphate-dependent enzyme and in addition has a depressed level of the nicotinamide adenine dinucleotide-dependent (catabolic) glutamate dehydrogenase, shows the same sensitivity to ammonium ion as the wild-type strain. These data suggest that the inhibition of amino acid transport by ammonium ion results from the uptake of this metabolite into the cell and its subsequent incorporation into the alpha-amino groups of glutamate and other amino acids.

Amino Acids

The effect of changes in pH on phosphate and potassium uptake by Monascus rubiginosus ATCC 16367 in submerged shaken culture.

Monascus rubiginosus ATCC 16367 was cultivated aerobically in media containing ammonium and nitrate as nitrogen source. The pH of the medium was adjusted at different times, the pH of the nitrate medium being lowered to the pH of the ammonium medium and the pH of the ammonium medium raised to that of the nitrate. More phosphate was taken up on the nitrate medium, but potassium uptake did not start until 24h. On the ammonium medium, both were taken up in parallel from the beginning, but the amount of phosphate taken up never reached the same level as on nitrate medium. When the pH was adjusted, the uptakes changed, especially on the ammonium medium where a great increase in phosphate uptake was observed. More conidia were formed on the nitrate medium and more pigment on the ammonium medium. When the pH of either media was adjusted, the development of conidia and pigment production changed to that of the other control medium where the pH evolved normally in the direction of the change, regardless of the source of nitrogen. The reasons for the development of conidia on nitrate medium or where the pH is high, and the production of pigment on ammonium medium or at low pH is discussed.

Ascomycota

[Changes in cephalic and peripheral use of glucose and glutamine under the influence of hyperammonemia in rats].

Arterio-venous differences of glucose and glutamine were determined across the brain and across the hind limb in normal and ammonium salt infused rats, before and during an insulin tolerance test, in an attempt to study the effect of hyperammonemia on cephalic and muscular metabolism. The results demonstrate that 1) hyperammonemia reduces the hind limb uptake of glucose without affect the cephalic uptake of glucose which is lowered during hypoglycemia, 2) the reduction of the cephalic and muscular glutamine output induced by the hypoglycemia is masked in presence of an hyperammonemia. In conclusion, it may be assume that, at the concentration obtained in this study, hyperammonemia does not act directly in the pathogenesis of hepatic coma in which a decrease in cerebral glucose uptake described; on the other hand, ammonium plays an important role in the muscle metabolism.

Ammonia

Ureidosuccinic acid permeation in Saccharomyces cerevisiae.

Some strains of Saccharomyces cerevisiae exhibit a specific transport system for ureidosuccinic acid, which is regulated by nitrogen metabolism. Ureidosuccinic acid uptake occurs with proline but with ammonium sulfate as nitrogen source it is inhibited. The V for transport is 20-25 mumol/ml cell water per min. The apparent Km is 3-10(-5) M. For the urep1 mutant (ureidosuccinic acid permease less) the internal concentration never exceeds the external one. In the permease plus strain ureidosuccinic acid can be concentrated up to 10 000 fold and the accumulated compound remains unchanged in the cells. Energy poisons such as dinitrophenol, carbonyl cyanide-m-chlorophenyldrazone (CCCP) or NaN3 inhibit the uptake. No significant efflux of the accumulated compound occurs even in the presence of these drugs. The specificity of the permease is very strict, only amino acids carrying an alpha-N-carbamyl group are strongly competitive inhibitors. The high concentration capacity of the cells and lack of active exit of the accumulated compound support the hypothesis of a carrier mediated active transport system.

Amino Acids