Uptake, metabolism and distribution of organic and inorganic nitrogen sources by Pinus sylvestris

Although an increasing number of studies show that many plant species have the capacity to take up amino acids from exogenous sources, the importance of such uptake for plant nitrogen nutrition is largely unknown. Moreover, little is known regarding metabolism and distribution of amino acid-N following uptake or of the regulation of these processes in response to plant nitrogen status. Here results are presented from a study following uptake, metabolism, and distribution of nitrogen from NO(3)(-) NH(4)(+), Glu, or Ala in Scots pine (Pinus sylvestris L). In a parallel experiment, Ala uptake, processing, and shoot allocation were also monitored following a range of pretreatments intended to alter plant C- and N-status. Uptake data, metabolite profiles, N fluxes through metabolite pools and tissues, as well as alanine aminotransferase activity are presented. The results show that uptake of the organic N sources was equal to or larger than NH(4)(+) uptake, while NO(3)(-) uptake was comparatively low. Down-regulation of Ala uptake in response to pretreatments with NH(4)NO(3) or methionine sulphoximine (MSX) indicates similarities between amino acid and inorganic N uptake regulation. N derived from amino acid uptake exhibited a rapid flux through the amino acid pool following uptake. Relative shoot allocation of amino acid-N was equal to that of NH(4)(+) but smaller than for NO(3)(-) Increased N status as well as MSX treatment significantly increased relative shoot allocation of Ala-N suggesting that NH(4)(+) may have a role in the regulation of shoot allocation of amino acid-N.     

Uptake of intact amino acids by plants depends on soil amino acid concentrations

Studies in different ecosystems have shown that plants take up intact amino acids directly but little is known about the influence of free amino acid concentrations in the soil on this process. We investigated the effect of three different soil amino acid N concentrations (0.025, 0.13 and 2.5 μg N g^?1 soil) on direct uptake of four dual labelled (¹⁵N, ¹³C) amino acids (glycine, tyrosine, lysine, valine) in a greenhouse experiment using Anthoxantum odoratum as a model plant.Our results revealed that 8–45% of applied ¹⁵N was incorporated into plant root and shoot tissue 48 h after labelling. Additional ¹³C enrichment showed that 2–70% of this incorporated ¹⁵N was taken up as intact amino acid. Total ¹⁵N uptake and ¹⁵N uptake as intact amino acids were significantly affected by soil amino acid N concentrations and significantly differed between the four amino acids tested.We found a positive effect of soil amino acid concentrations on uptake of mineralized ¹⁵N relative to amino acid concentrations for all amino acids which was presumably due to higher diffusion rates of mineralized tracer to the root surface. However, intact amino acid uptake relative to amino acid concentrations as well as the proportion of total ¹⁵N taken up directly decreased with increasing soil amino acid N concentrations for all amino acids, irrespective of their microbial degradability. This effect is most likely controlled by the mineral N concentration in soil and perhaps in plants which inhibits direct amino acids uptake.Overall, we conclude that plant internal regulation of amino acid uptake controlled by mineral N is the main mechanism determining direct uptake of amino acids and thus a lower contribution of intact amino acid uptake to the plants N nutrition has to be expected for higher amino acid concentrations accompanied by mineralization in soil.     

Regulation of amino acid uptake in conifers by exogenous and endogenous nitrogen

Although an accumulating amount of research clearly indicates that plants are capable of taking up exogenous amino acids, the actual importance of such organic N sources for plant N nutrition is under debate. In this study, we show that amino acid uptake by Scots pine (Pinus sylvestris L.) is significantly decreased by elevated internal NH(4)(+) levels, while it increases following exposure to exogenous amino acids. Furthermore, amino acid uptake is larger in N-deficient plants than in plants grown with a large access of N. The regulatory pattern of amino acid uptake shows important similarities to the regulation of NO(3)(-) and NH(4)(+) transport as well as to the regulation of yeast amino acid transporters. In addition, our data suggest that uptake may be regulated by factors not originating from N metabolism. The up-regulation of uptake in response to N deficiency suggests that amino acid uptake may be a significant contributor to the N economy of P. sylvestris.     

Glucose-induced Release of Amino Acids from Saccharomyces carlsbergensis by Action on the Cytoplasmic Membrane

When washed yeast cells grown under appropriate conditions were suspended in glucose solution there was a sudden release of α-amino nitrogen into the medium. This released material was of low molecular weight, and its composition was closely similar to that of the intracellular free amino acid pool. During the leakage of amino acids, the yeast did not efficiently absorb labeled amino acids added to the test medium, despite the rapid uptake and metabolism of glucose. Uptake of a labeled amino acid and reabsorption of the released α-amino nitrogen occurred almost simultaneously. When these yeast cells were exposed to glucose in the presence of calcium ions, leakage was strongly inhibited. Butanol under the same conditions increased glucose-induced leakage of cell contents. The adenosine triphosphatase activity of intact yeast cells exposed to glucose was greater than that of cells exposed to water. Yeast cells treated with glucose prior to equilibration with sorbose exhibited less ability to retain the sorbose when washed at 0 C than did cells pretreated with water. It was concluded that glucose-induced leakage of amino acids was the result of two factors acting together. These were (i) a change in membrane permeability associated with glucose uptake, and (ii) a temporary shortage of energy for amino acid uptake or retention.     

Uptake and assimilation of nitrogen from solutions containing multiple N sources

We assessed the extent to which plants can acquire amino acids when supplied as single N-sources or when plants have access to a mixture of amino- and inorganic N sources. Because the uptake of different N-sources is temperature-dependent, the effects of temperature on amino-N uptake were also tested. Lolium perenne (perennial rye-grass) was grown hydroponically at 11 °C or 21 °C. Uptake of N was determined using ¹⁵N tracers at the growth temperature from solutions containing either nitrate, ammonium or glycine as single N sources and from a mixture containing all three N-forms. Estimates of the relative importance of amino acids such as glycine to the total N budget of plants will have been underestimated in studies where uptake was determined in single source solutions compared with those from solutions containing a mixture of N-forms. The proportion of total N acquired from the mixed N source as ammonium increased as temperature was reduced. Regarding the uptake and initial metabolism of glycine, uptake was probably the rate limiting step at 11 °C whilst it was the metabolism of glycine to serine at 21 °C. Although ¹⁵N incorporation into the plant amino-N pool was generally in proportion to the abundance of individual amino acids, its incorporation into the glycine pool was sometimes significantly less than predicted.     

Uptake of the neutral amino acids glutamine, leucine, and serine by Pneumocystis carinii.

Experiments to elucidate the mechanism by which Pneumocystis carinii transports glutamine, leucine, and serine were performed in this study. Uptake of all three radiolabeled amino acids exhibited first-order, saturation kinetics as extracellular substrate concentrations were increased, thus ruling out simple diffusion and indicating carrier-mediated transport. Kinetic analyses of amino acid uptake and the results of competitive inhibition experiments suggested that leucine, serine, and glutamine were taken up via a common transporter system. The uptake of serine was examined in greater detail to characterize the nature of the carrier. Serine uptake was not affected by N, N'-dicyclohexylcarbodiimide, carbonyl cyanide m-chlorophenyl hydrazone, ouabain, gramicidin, valinomycin, sodium azide, salicylhydroxamine acid (SHAM), iodoacetate, iodoacetate plus SHAM, KCN, and azide. Thus serine uptake did not require sodium or energy from ATP, an electrochemical proton gradient or a membrane potential across the cell surface (i.e., proton-motive force). Serine uptake was dependent on glucose in the extracellular compartment. In the presence of glucose, serine uptake was inhibited by chloramphenicol but not cycloheximide. The results from these experiments are most consistent with facilitated diffusion as the mechanism. After 30 min of incubation, most of the radioactivity was in the cellular soluble fraction. In most cases, incorporation into the extractable total lipids and the remaining particulate cellular components were detectable after this incubation period.     

Carbohydrate Effects on Amino Acid Transport by Trypanosoma equiperdum*

SYNOPSIS. Uptake of ¹⁴C-labeled alanine, glutamate, lysine, methionine, proline, and phenylalanine by Trypanosoma equiperdum during 2-minute incubations occurred by diffusion and membrane-mediated processes. Amino acid metabolism was not detected by paper chromatography of trypanosome extracts. Most of 18 carbohydrates tested for ability to alter amino acid transport neither changed nor significantly inhibited transport. Glucose, however, stimulated glutamate, lysine and proline transport; fructose stimulated lysine uptake and 2-deoxy-D-glucose increased phenylalanine and methionine absorption. No evidence was found that the carbohydrates acted by binding to amino acid transport “sites.” Glucose inhibition of alanine, phenylalanine, and methionine uptake was linked to glycolysis. The rapid formation of alanine from glucose stimulated alanine release and, when glycolysis was blocked, glucose no longer inhibited alanine transport. Methionine and phenylalanine release was also stimulated by glucose. Glucose changed the ability of lysine, glutamate, and proline to inhibit each others’uptake, indicating that certain amino acids are preferentially absorbed by respiring cells. Analysis of free pool amino acid levels suggested that some amino acid transport systems in T. equiperdum are linked in such a way to glycolysis as to control the cell concentrations of these amino acids.     

Post-uptake metabolism affects quantification of amino acid uptake

? The quantitative significance of amino acids to plant nutrition remains controversial. This experiment determined whether post-uptake metabolism and root to shoot export differ between glycine and glutamine, and examined implications for estimation of amino acid uptake. ? Field soil containing a Eucalyptus pauciflora seedling was injected with uniformly (13)C- and (15)N-labelled glycine or glutamine. I quantified (15)N and (13)C excess in leaves and roots and intact labelled amino acids in leaves, roots and stem xylem sap. A tunable diode laser quantified fluxes of (12)CO(2) and (13)CO(2) from leaves and soil. ? 60-360 min after addition of amino acid, intact molecules of U-(13)C,(15)N glutamine were < 5% of (15)N excess in roots, whereas U-(13)C,(15)N glycine was 30-100% of (15)N excess in roots. Intact molecules of glutamine, but not glycine, were exported from roots to shoots. ? Post-uptake metabolism and transport complicate interpretation of isotope labelling such that root and shoot contents of intact amino acid, (13)C and (15)N may not reflect rates of uptake. Future experiments should focus on reconciling discrepancies between intact amino acid, (13)C and (15)N by determining the turnover of amino acids within roots. Alternatively, post-uptake metabolism and transport could be minimized by harvesting plants within minutes of isotope addition.     

Amino acid uptake by Ricinus communis roots: characterization and physiological significance

Abstract Roots of sterile-grown, intact 6-day-old seedlings of Ricinus communis possess at least two independent active amino acid uptake systems, one for neutral and one for basic amino acids. The kinetics of uptake of L-proline and L-arginine, which were taken as representative substrates for the two systems, are biphasic. At low concentrations (0.01–0.5 mol m^?3) Michaelis -Menten kinetics prevail, changing to a linear concentration dependence at higher substrate concentrations (1–50 mol m^?3). L-glutamate uptake velocity is linear over the whole substrate concentration range. For comparison the uptake kinetics of nitrate and ammonium were determined as well as interactions among the different nitrogen sources. The K_m value for nitrate uptake was 0.4 mol m^?3, and for ammonium 0.1 mol m^?3. The uptake capacity for nitrate or ammonium was approximately the same as for amino acids. The interaction between the uptake systems for organic and inorganic nitrogen is small. Two hypotheses for the physiological significance of amino acid uptake by roots were considered: (i) Uptake of amino acids from the soil-determination of amino acids in soil and in soil water indicates that they might contribute 15–25% to the nitrogen nutrition of the plant. (ii) Amino acid uptake systems of root cells serve primarily as retrieval of amino acids delivered from the phloem- it was found that ¹⁴C L-glutamine, which was delivered to the cotyledon and transported to the root via the phloem, was not lost by the roots, whereas it appeared in the bathing medium if L-glutamine was applied externally to the root to compete for the uptake sites; this suggests that an apoplastic pool of amino acids in the root exists due to their efflux from the phloem.     

Solute transport into healthy and powdery mildew-infected leaves of pea and uptake by powdery mildew mycelium

The transport of sugars and amino acids into the mycelium of Erysiphe pisi DC. was investigated using two different systems, intact leaf discs and mycelial suspensions. Of the sugars tested, glucose was preferentially taken up by both uninfected and mildew-infected leaf discs, whereas glutamine was taken up by both tissues at a higher rate than lysine or aspartic acid. Leaf discs from infected tissue had a greater uptake capacity than those from healthy tissue for both sugars and amino acids. The uptake of glucose was inhibited more markedly than that of sucrose and fructose by 10 μ m carbonyl cyanide m -chlorophenylhydrazone (CCCP), 1 m m N -ethylmaleimide (NEM), 1 m m diethyl pyrocarbonate (DEPC) and 1 m m phenylglyoxal, whereas 1 m m PCMBS ( p -chloro-mercuribenzenesulphonic acid) inhibited sucrose uptake to the greatest extent. Uptake of glutamine, lysine and aspartic acid was inhibited similarly by CCCP (80%), NEM (20%), DEPC (70%) and PCMBS (60%). Additionally, leaf discs were used to determine which solutes could be taken up from leaf tissue by the fungus. The uptake of sugars into the mycelium was greater than that of amino acids.
Suspensions of powdery mildew mycelium accumulated glucose at about three times the rate of sucrose or fructose, and the amino acid glutamine was taken up at three times the rate of lysine or aspartic acid. Spores separated from the suspension had a low uptake capacity.
When the reducing sugar concentration of leaf apoplastic fluid was estimated, leaves infected by powdery mildew had much higher amounts in the apoplast, whereas the activity of acid invertase also appeared to be higher in apoplastic fluids from infected leaves. When apoplastic fluid samples were run on SDS gels, an invertase antibody detected two bands in samples from infected tissues that were not found in the uninfected samples.     

Isolation and characterization of a general amino acid permease from the ectomycorrhizal fungus Amanita muscaria

A cDNA coding for a fungal amino acid transporter ( AmAAP1 ) was identified from Amanita muscaria ectomycorrhizas. The transporter gene was expressed at a basal level under all conditions investigated, but its expression was enhanced 10-fold in the absence of a N source utilized by the fungus. Nitrate was not a suitable N source for A. muscaria and resulted in maximal AmAAP1 expression. The expression of AmAAP1 in a yeast mutant revealed its function as a high-affinity amino acid transporter with a broad substrate spectrum. AmAAP1 takes up all investigated amino acids with K _m values between 22 μM for histidine and up to 100 μM for proline. Gene expression and amino acid uptake data together indicate two main functions for AmAAP1: uptake of amino acids from soil for fungal nutrition, and prevention of an amino acid loss by hyphal leakage in the absence of a suitable N source.     

Resistance of soil protein depolymerization rates to eight years of elevated CO<Subscript>2</Subscript>, warming,and summer drought in a temperate heathland

Soil N availability for plants and microorganisms depends on the breakdown of soil polymers such as proteins into smaller, assimilable units by microbial extracellular enzymes. Changing climatic conditions are expected to alter protein depolymerization rates over the next decades, and thereby affect the potential for plant productivity. We here tested the effect of increased CO₂ concentration, temperature, and drought frequency on gross rates of protein depolymerization, N mineralization, microbial amino acid and ammonium uptake using ¹⁵N pool dilution assays. Soils were sampled in fall 2013 from the multifactorial climate change experiment CLIMAITE that simulates increased CO₂ concentration, temperature, and drought frequency in a fully factorial design in a temperate heathland. Eight years after treatment initiation, we found no significant effect of any climate manipulation treatment, alone or in combination, on protein depolymerization rates. Nitrogen mineralization, amino acid and ammonium uptake showed no significant individual treatment effects, but significant interactive effects of warming and drought. Combined effects of all three treatments were not significant for any of the measured parameters. Our findings therefore do not suggest an accelerated release of amino acids from soil proteins in a future climate at this site that could sustain higher plant productivity.     

Characterization of tryptophan transport in human placental brush-border membrane vesicles.

The characteristics of tryptophan uptake in isolated human placental brush-border membrane vesicles were investigated. Tryptophan uptake in these vesicles was predominantly Na+-independent. Uptake of tryptophan as measured with short incubations occurred exclusively by a carrier-mediated process, but significant binding of this amino acid to the membrane vesicles was observed with longer incubations. The carrier-mediated system obeyed Michaelis-Menten kinetics, with an apparent affinity constant of 12.7 +/- 1.0 microM and a maximal velocity of 91 +/- 5 pmol/15 s per mg of protein. The kinetic constants were similar in the presence and absence of a Na+ gradient. Competition experiments showed that tryptophan uptake was effectively inhibited by many neutral amino acids except proline, hydroxyproline and 2-(methylamino)isobutyric acid. The inhibitory amino acids included aromatic amino acids as well as other system-1-specific amino acids (system 1 refers to the classical L system, according to the most recent nomenclature of amino acid transport systems). The transport system showed very low affinity for D-isomers, was not affected by phloretin or glucose but was inhibited by p-azidophenylalanine and N-ethylmaleimide. The uptake rates were only minimally affected by change in pH over the range 4.5-8.0. Tryptophan uptake markedly responded to trans-stimulation, and the amino acids capable of causing trans-stimulation included all amino acids with system-1-specificity. The patterns of inhibition of uptake of tryptophan and leucine by various amino acids were very similar. We conclude that system t, which is specific for aromatic amino acids, is absent from human placenta and that tryptophan transport in this tissue occurs via system 1, which has very broad specificity.     

Regulation of amino acid transport in L6 muscle cells: I. Stimulation of transport system a by amino acid deprivation

The regulation of amino acid transport in L6 muscle cells by amino acid deprivation was investigated. Proline uptake was Na⁺-dependent, saturable and concentrative, and was predominantly through system A. Proline uptake was inhibited by alanine, α-amino isobutyric acid (AIB), and by α-methylamino isobutyric acid, but not by lysine or valine. At 25°C, Km of proline uptake was 0.5 mM. Amino acid-deprivation resulted in a progressive increase in the rate of proline uptake, reaching up to 6-fold stimulation after 6 hours. The basal and stimulated transport were equally Na⁺-dependent, and both were inhibited by competition with the same amino acids. Kinetic analysis showed that Km decreased by a factor of 2.4 and Vmax increased 1.9-fold in deprived cells. Amino acid-deprivation did not stimulate amino acid uptake through systems other than system A. This suggests that the higher Km in proline-supplemented cells is not due to release of intracellular amino acids into unstirred layers surrounding the cells. The presence of amino acids which are substrates of system A (including AIB) during proline-deprivation, prevented stimulation of proline uptake, whereas those transported by systems Ly⁺ or L exclusively were ineffective. The stimulation of the transport-rate in deprived cells could be reversed by subsequent exposure to proline or other substrates of system A. L6 cells, deprived of proline for 6 hours, retained the stimulation of transport after detachment from the monolayers with trypsin. Uptake rates were comparable in suspended and attached cells in monolayer culture. Thus, amino acid-depreivation of L6 cells results in an adaptive increase in proline uptake, which is not due to unstirred layers but appears to be mediated by other mechanisms of selective transport regulation.     

Effect of glucocorticoides on the release of amino acids in the perfused rat hindquarter (author's transl)]

The release of amino acids by skeletal muscle was studied in the isolated perfused rat hindquarter. Adrenalectomy depressed the formation of glutamine and alanine as well as the efflux of all other amino acids measured. Betamethasone--a synthetic glucocorticoid--caused a significant increase in the efflux of nearly all amino acids up to the level of normal controls. The release of amino acids was also increased in perfused hindquarters of diabetic rats. On the other hand, insulin exhibited a depressing effect on the release of amino acids by hindquarters of normal rats. The metabolic integrity of the muscle tissue was proved by measuring creatine phosphate, ATP, ADP and water content as well as by the significant insulin effect on glucose uptake and on [14C]leucine incorporation into muscle proteins.     

l-[3H]Nitroarginine and l-[3H]Arginine Uptake into Rat Cerebellar Synaptosomes: Kinetics and Pharmacology

Abstract: Characteristics of the transport of the nitric oxide synthase substrate l -arginine and its inhibitor, N ^G-nitro- l -arginine ( l -NOARG), into rat cerebellar synaptosomes were studied. Uptake of both l -arginine and l -NOARG was linear with increasing amount of protein (up to 40 µg) and time of incubation (up to 5 min) at 37°C. Uptake of both compounds reached a steady state by 20 min. Maximal uptake of l -NOARG (650 pmol/mg of protein) was three to four times higher than that of l -arginine (170 pmol/mg of protein). l -NOARG uptake showed biphasic kinetics ( K _{m 1} = 0.72 m M , V _{max 1} = 0.98 nmol/min/mg of protein; K _{m 2} = 2.57 m M , V _{max 2} = 16.25 nmol/min/mg of protein). l -Arginine uptake was monophasic with a K _m of 106 µ M and a V _max of 0.33 nmol/min/mg of protein. l -NOARG uptake was selectively inhibited by l -NOARG, N ^G-nitro- l -arginine methyl ester, and branched-chain and aromatic amino acids. l -Alanine and l -serine also inhibited l -NOARG uptake but with less potency. Uptake of l -arginine was selectively inhibited by N ^G-monomethyl- l -arginine acetate and basic amino acids. These studies suggest that in rat cerebellar synaptosomes, l -NOARG is transported by the neutral amino acid carrier systems T and L with high affinity, whereas l -arginine is transported by the basic amino acid carrier system y⁺ with high affinity. These data indicate that the concentration of competing amino acids is an important factor in determining the rates of uptake of l -NOARG and l -arginine into synaptosomes and, in this way, may control the activity of nitric oxide synthase.     

Some Factors Which Affect Amino Acid Uptake by Saccharomyces carlsbergensis

When fully grown cells of Saccharomyces carlsbergensis were suspended in a solution of glucose and labeled amino acids, there was a lag phase before rapid uptake of certain amino acids. During this lag, significant amounts of sugar were utilized. The lag phase varied in length, depending upon the amino acid under study, but could be shortened by aeration of the cells and eliminated by their preincubation in glucose solution. Divalent metal ions, especially Ca²⁺ added during the early stages of the lag phase, increased the length of the lag, an effect that could be reversed by washing with ethylenediaminetetraacetate, but amino acids which normally showed little or no lag before uptake were insensitive to Ca²⁺. The rate of uptake of amino acids or of sugar was essentially unaffected by Ca²⁺, whereas 2,4-dinitrophenol caused an overall decrease in the rate of uptake of all amino acids tested. The relevance of these observations to commercial brewing practice is shown.     

Effect of Dipeptides on the Growth of <Emphasis Type="Italic">Oenococcus oeni</Emphasis> in Synthetic Medium Deprived of Amino Acids

Oenococcus oeni has numerous amino acid requirements for growth and dipeptides could be important for its nutrition. In this paper the individual or combined effect of dipeptides on growth of O. oeni X₂L in synthetic media deficient in one or more amino acids with L-malic acid was investigated. Utilization of dipeptides, glucose, and L-malic acid was also analyzed. Dipeptides were constituted by at least one essential amino acid for growth. Dipeptides containing two essential amino acids, except leucine, had a more favorable effect than free amino acids on the growth rate. Gly-Gly was consumed to a greater extent than Leu-Leu and a rapid exodus of glycine to the extracellular medium accompanied it. The microorganism could use glycine in exchange for other essential amino acids outside the cell, favoring growth. In the presence of Leu-Leu, the increase in glucose consumption rate could be related to the additional energy required for dipeptide uptake.