Studies in the Physiology of Lichens: With one Figure in the Text: Absorption and Utilization of Some Simple Organic Nitrogen Compounds by Peltigera polydactyla

Peltigera discs can accumulate relatively very large amountsof asparagine from solution by a process which involves theentry and accumulation of asparagine in the tissues as the completemolecule. Utilization of absorbed asparagine occurs at a relativelyslow rate and involves the preliminary deamidation of it toammonia. Asparagine absorption is completely inhibited by sodiumfluoride, and partially inhibited by glucose. Absorption isaccompanied by a release of ammonia in the medium and also byincreases in respiration rates and a change in R.Q. None ofthe absorbed asparagine can be washed out of the tissues. Discsalso show strong powers of absorption from solutions of glutamine,and glutamic and aspartic acids. There is more organic thaninorganic nitrogen in solution in the liquids available to thelichen in its habitat. The amounts of glucose and of phosphate which Peltigera andsome other lichens can accumulate from solution are also verylarge considering the slow growth-rates of these plants. Itis suggested that the possession of strong powers of absorptionof substances from solution may be a general characteristicof lichens and plants of similar habitats: it may representan adaptation to existence in barren conditions where the concentrationof nutrients in solution is very low. Utilization of absorbednutrients at a slow rate may be an adaptation to the need tosurvive long periods of starvation. The ecological implicationsof these suggestions are also discussed.     

The Effect of Molybdenum upon the Nitrogen Metabolism of Anabaena cylindrica: II. A More Detailed Study of the Action of Molybdenum in Nitrate Assimilation

Molybdenum-deficiency in Anabaena cylindrica results in a decreaseof all the organic nitrogen fractions determined analyticallywith the exception of amide which occurs at approximately thesame concentration irrespective of the molybdenum status. Molybdenum-rich,nitrogen-starved cells assimilate nitrate to protein rapidlyin the dark; similarly treated molybdenum-deficient cells rapidlyreduce nitrate to ammonia and amide, but the further synthesisto peptides and proteins proceeds very slowly. The rate of endogenousrespiration is higher in deficient than in normal cells, butthe rate of glycolysis is lower. No period of adaptation isrequired, after the addition of molybdenum to deficient cells,before normal nitrogen metabolism commences. Glucose, acetate,fumarate, succinate, and citrate are respired by normal anddeficient cells in the dark. Acetate has no effect on nitratereduction, glucose stimulates the reduction for a short period,while fumarate (and probably succinate) and, somewhat less efficiently,citrate, stimulate reduction in both normal and deficient cellsand enable the deficient cells to assimilate nitrate completelyto protein. It would seem that, in the absence of molybdenum,there is an insufficient supply of hydrogen and/or energy donorsto permit complete nitrate assimilation.     

Changes in non-protein nitrogen metabolism during tobacco mosaic virus biosynthesis

1. Discs cut from tobacco leaf tissue infected with tobacco mosaic virus and cultured in water contain less non-protein nitrogen than comparable uninfected discs during the time at which TMV is formed. This deficiency disappears when virus formation ceases. Discs cultured in nutrient solution form about twice as much TMV as discs cultured in water. The maximum non-protein nitrogen deficiency is comparable in magnitude to the amount of virus synthesized. 2. The largest difference between injected and uninfected tissue occurs in the ammonia content. Smaller, but significant differences in amide content are found. Infected discs cultured in water show no significant differences from control discs in free amino acid content; infected discs cultured in nutrient solution develop a small deficiency in amino acid nitrogen. 3. The general patterns of change in composition of the pool of soluble nitrogen are similar in both infected and uninfected discs. 4. The data indicate that the bulk of the nitrogen incorporated into virus protein is withdrawn from the leaf's pool of soluble nitrogen; virus is formed de novo from ammonia nitrogen and non-nitrogenous carbon sources. The effect of virus infection on host nitrogen metabolism appears to be due to the formation of virus rather than to its presence.     

Utilization of Nitrogen Sources by Immature Soybean Cotyledons in Culture

Immature Glycine max (L.) Merrill cotyledons were cultured ina defined medium containing different nitrogen sources. Glutaminewas the most efficient source in terms of protein accumulationin the cotyledons. Asparagine was less efficient (about 70 percent that of glutamine) while allantoin was a poor source ofnitrogen. This was also true for older cotyledons where asparaginaseand allantoinase activities were maximal. The utilization ofboth asparagine and allantoin (but not glutamine) was totallyinhibited by methionine sulfoximine suggesting that their metabolisminvolves ammonia assimilation via glutamine synthetase. Apparently,neither exogenous or endogenously-generated ammonia had mucheffect on glutamine utilization, but ammonia did have a smallinhibitory effect on asparagine, which may in part account forthe lower efficiency observed with this amide. Glycine max, soybean, cotyledon culture, nitrogen metabolism     

Nitrogen Metabolism of Detached Kalanchoe Leaves in the Dark in Relation to Acidification, Deacidification, and Oxygen Uptake

Detached Kalancho leaves were placed in the dark and changesin the amounts of total soluble N, asparagine, glutamine, aminoN, ammonia, and titratable acid were followed during the periodsof acidification and deacidification. Abundant starch was presentthroughout the experiments. The amount of acid which accumulatedin the initial period was increased by placing the leaves in5 per cent. CO₂ and also by decreasing the temperature from25 to 10°. In neither case were the nitrogen fractions affectedin a similar way to the acid levels. No consistent relationshipcould be found between the amount or change in amount of acidand the amounts of the nitrogen fractions. Over prolonged periodsthe drifts in the amounts of the nitrogen fractions were verysimilar to those known in non-succulent leaves. It is concludedthat, despite the large amounts of free acid and starch present,the nitrogen metabolism of detached Kalancho leaves in the darkis similar to that of non-succulent leaves. A possible explanationis suggested for the incorporation of ¹⁴C from ¹⁴CO₂ into nitrogenoussubstances in Kalancho leaves during dark acidification. Changesin the amounts of glutamine, which seemed to be related to oxygenuptake rather than to acid fluctuations, were measured in comparableleaf samples showing different rates of oxygen uptake. The rateof oxygen uptake of leaves and leaf disks in the dark was controlledby placing them in an atmosphere of nitrogen, or 5, 20, or 100per cent, oxygen, by the addition of 10^–3 M. cyanide,or by varying the temperature. At the end of a given periodthe amount of glutamine was always greater in the samples whichhad shown the higher rate of oxygen uptake during this perod.This correlation was found irrespective of whether the amountof glutamine had increased or decreased. With the assumptionsthat glutamine production and consumption were proceeding simultaneouslyand that the rate of consumption was unaffected by the rateof oxygen uptake, the experimental values are consistent withthe hypothesis that the rate of glutamine production and therate of oxygen uptake were directly related. The possible significanceof these findings is discussed.     

The Assimilation of Ammonia by Nitrogen-starved Cells of Chlorella vulgaris: Part I. The Correlation of Assimilation with Respiration

The addition of ammonium sulphate to a suspension of nitrogen-starvedChlorella cells is followed immediately by the rapid assimilationof ammonia and a large increase of the respiration rate. Theassimilation of ammonia and the high rate of respiration continueuntil either all the ammonia has been assimilated or some carbonreserve within the cells has been exhausted. Which happens firstdepends on the amount of ammonia added and the quantity of cellspresent. The respiration which accompanies, ammonia assimilationis sensitive to cyanide and it has a respiratory quotient of0•75 compared with 1•2–1•3 for normal reapiration.The addition of glucose to nitrogen-starved cells when ammoniais being assimilated does not increase either the rate of respirationor the rate of assimilation. The rates of reapiration and ammoniaassimilation by normal cells are markedly increased by the additionof glucose. Light has little effect on the rate of ammonia assimilationby nitrogen-starved cells, but doubles the assimilation rateof normal cells.     

The Movement of Organic Nitrogen Compounds in Plants: With two Figures in the Text

The hypothesis that the movement of organic nitrogen, like thatof carbohydrate, is governed by the prevailing concentrationgradients for the mobile compounds in the phloem has been testedby determining the effect on nitrogen distribution when normaltranslocation was interrupted by ringing in Pelargonium spp.and Vicia faba plants. Nitrogen compounds in the plant tissue were fractionated intoprotein, soluble protein, amide, amino-acid, peptide, nitrate,and ammonia nitrogen. The presence of short-chain peptides inthe extracts was confirmed by paper chromatography. Results showed that accumulation above the ringed zone was confinedto the soluble nitrogen fractions and that amino-acids and possiblypeptides were involved in translocation. The direction of movementmay be determined by the prevailing concentration gradientsof these compounds in the phloem tissue.     

Studies in the Physiology of Lichens: Experiments with Dissected Discs of Peltigera polydactyla

The medulla of Peltigera discs can be separated by dissectionfrom the overlying region which comprises the algal layer andupper cortex. In most cases, dissection has no marked effectupon the isolated tissue zones, so that a comparison of themgives a good approximate indication of the relative behaviourof the corresponding regions of the intact thallus. The behaviourof the medulla, entirely fungal in composition, gives some indicationof the physiology of the lichen fungus. The algal region has a greater respiration rate and nitrogencontent than the medulla, and it absorbs more glucose, asparagine,phosphate, and ammonia than the medulla. Compared to the medulla,the algal region absorbs more glucose than asparagine and thisis attributed to a marked preference for glucose on the partof the algal cells. The medulla has a greater water contentthan the algal region, and may function as a water reservoir.     

Energy metabolism in the digestive tract and liver of cattle: influence of physiological state and nutrition

Major functions of portal-drained viscera (PDV) and liver of cattle include absorption of digestion products and modification of the body's supply of intermediary metabolites. The disproportionately high metabolic rate of PDV and liver (7-13% of body tissues) is exemplified by their oxygen uptake (40-50% of whole body). Extensive metabolism of glucose, volatile fatty acids and amino acids by PDV modulates nutrient supply from the diet such that most responses to diet or physiological state are a function of level of diet intake. Similarly, blood flow through PDV is highly correlated with energy intake across a range of body weight, physiological state or diet composition. Most common dietary responses in metabolite uptake by PDV are changes in uptake of ammonia and volatile fatty acids, which emphasize the strong energy: nitrogen interrelationship in the rumen and subsequently the rest of the body. The liver (tissue in series with PDV) removes glucose precursors and ammonia from its blood supply as part of its functions in gluconeogenesis, ammonia detoxification and urea synthesis. The liver also alters amounts and proportions of amino acids supplied by PDV. Accountable percentages of metabolizable energy from net PDV supply include: organic acids, 41-59%; amino acids, 5-13%; and heat energy (from oxygen uptake), 11-22%.     

Changes in the Nitrogen Compounds of Xylem Sap of Mulberry (Morus alba L.) during Regrowth after Pruning

Changes in fresh and dry weights and total nitrogen in stemsections of mulberry (Morus alba L.) and seasonal fluctuationin the amounts of exudate, pH and nitrogen compounds in xylemsap from cut stems of the plant after pruning have been studied.The amount and concentrations of nitrogen compounds in the sapchanged during the experimental period, but nitrate-nitrogenand ammonia-nitrogen were constant constituents, and asparaginewas quantitatively the major organic nitrogen compound. Smalleramounts of glutamine, aspartic acid and glutamic acid were alsofound, but no ureides or alkaloids were detected. Relationshipsbetween growth and nitrogen in xylem sap of mulberry and therole and importance of asparagine in nitrogen metabolism arediscussed. Morus alba L., mulberry, asparagine, exudates, nitrogen, amino-acids, xylem sap, stem growth, aspartic acid, glutamic acid, ureides     

Sugar Absorption and Surface Carbohydrase Activity of Peltigera polydactyla (Neck.) Hoffm.

A method suitable for the study of the physiology of absorptionof substances from solution and of respiration of Peltigerapolydactyla was elaborated making use of discs of tissue whichcould be floated on solutions. The rate of absorption of hexosesugars by the discs was sensitive to temperature, the presenceof inhibitors, such as sodium fluoride and silver nitrate, butrelatively insensitive to hydrogen ion concentration. Glucosewas absorbed preferentially to fructose from equimolar solutions.Absorption of sugar from sucrose solutions involved inversionby an enzyme system held on the cell surface. This system wasrelatively unaffected by exposure to moderate temperature, tosodium fluoride or silver nitrate but sensitive to hydrogenion concentration. Sucrose did not seem to be absorbed as suchand the glucose was absorbed preferentially from the productsof inversion. All parts of the thallus were equally effectivein inversion but absorption was mainly into the algal layer.The entry into the tissues was associated with a respiratoryrise.     

Metabolism of urea in Chlorella ellipsoidea

The effect of cyanide on ammonia and urea metabolism was studiedwith intact cells of Chlorella ellipsoidea Gerneck, a greenalga which apparently lacks urease. Ammonia uptake was inhibited more readily by cyanide than wasurea uptake. Urea uptake was stimulated by lower concentrationsof cyanide. The addition of cyanide caused the formation ofammonia from some cellular nitrogenous compounds. In the presenceof exogenously added urea, the molar ratio of ammonia accumulatedin the medium to urea taken up exceeded 2.0 as the cyanide concentrationincreased. However, the molar ratio of ammonia actually producedfrom urea nitrogen to urea taken up was less than 1.35 at anyconcentration of cyanide tested. In the presence of higher concentrationsof cyanide, the rate of incorporation of ¹⁵N into amino acidsfrom ¹⁵N-urea was higher than that from ¹⁵N-ammonium sulfate. The results suggest that Chlorella ellipsoidea possesses a pathwaythrough which urea nitrogen is assimilated directly withouta preliminary breakdown to ammonia. (Received October 18, 1976; )     

Amino Acid metabolism in pea leaves : utilization of nitrogen from amide and amino groups of [N]asparagine

The flow of nitrogen from the amino and amide groups of asparagine has been followed in young pea (Pisum sativum CV Little Marvel) leaves, supplied through the xylem with ¹⁵N-labeled asparagine. The results confirm that there are two main routes for asparagine metabolism: deamidation and transamination.

Nitrogen from the amide group is found predominantly in 2-hydroxy-succinamic acid (derived from transamination of asparagine) and in the amide group of glutamine. The amide nitrogen is also found in glutamate and dispersed through a range of amino acids. Transfer to glutamineamide results from assimilation of ammonia produced by deamidation of both asparagine and its transamination products: this assimilation is blocked by methionine sulfoximine. The release of amide nitrogen as ammonia is greatly reduced by aminooxyacetate, suggesting that, for much of the metabolized asparagine, transamination precedes deamidation.

The amino group of asparagine is widely distributed in amino acids, especially aspartate, glutamate, alanine, and homoserine. For homoserine, a comparison of N and C labeling, and use of a transaminase inhibitor, suggests that it is not produced from the main pool of aspartate, and transamination may play a role in the accumulation of homoserine in peas.

     

Ammonia production from amino acids and urea in the caecal contents of the chicken

1. Ammonia production from urea and amino acids in the caecal contents of the chicken was evaluated using 15N-labeled nitrogenous compounds. 2. About 43% of each of urea nitrogen and glutamine amide nitrogen was converted to ammonia nitrogen, but only 25% of epsilon-nitrogen of the added arginine, a precursor of urea, was found in ammonia. 3. Amino nitrogen of the separately added glutamic acid and glycine to be converted to ammonia was 19-20% of their added amounts, whereas that of alpha-alanine was 11%. 4. It is concluded that dietary and urinary amino acids and urea which find their ways into the caeca are useful nitrogen sources for ammonia production by microflora in the caeca of the chicken.     

Studies on the Growth in Culture of Plant Cells: VII. EFFECTS OF KINETIN ON THE CARBOHYDRATE AND NITROGEN METABOLISM OF ACER PSEUDOPLATANUS, L CELLS GROWN IN SUSPENSION CULTURE

Aspects of the carbohydrate and nitrogen metabolism of Acerpseudcplatanus, L cell suspension cultures grown on a syntheticmedium containing 2 per cent glucose and 1.0 mg/l 2,4-dichlorophenoxyaceticacid and kinetin either at 0.25 mg/l (low kinetin) or at 2.5mg/l (high kinetin) are described. High kinetin inhibits growthas measured by increase in cell number, packed-cell volume,and cell dry weight. Although not inhibitory to glucose utdization,high kinetin inhibits the O₂ uptake of the cells. Such cellscontain only a trace amount of fructose and their rate of O₂uptake can be raised to that of the low kinetin cells by a periodof fructose feeding. The O₂ uptake of both kinds of cell issensitive to malonate but the stimulation of O₂ uptake inducedby bis(hexafiuoroacetonyl)-acetone (‘1799’) at 0.2mM is much less with the high-kinetin than the low-kinetin cells.The enzymes phosphoglucoseiseomerase and glucose-6-phosphatedehydrogenase are much less active in the high-kinetin cells.Mitochondria isolated from both kinds of cells show good respiratorycontrol although slightly lower values for Q_O2(N), ADP/O ratioand control ratio are recorded with mitochondria from the highkinetin cells. Kinetin at 2.5 mg/l slightly reduces the ADP/Oratio of isolated mitochondria but at 4.0 mg/l their responseto ADP is completely suppressed. Extracellular hemicelluloseformed in presence of high kinetin has a reduced content ofgalactose and xylose and an increased content of glucose. Theseobservations indicate that the inhibition of respiration byhigh kinetin is mainly due to suppression of glucose conversionto other sugars rather than to inhibition of glycolysis or terminalrespiration. High kinetin decreases the rate of protein but not of amino-acidsynthesis. Suppression of the synthesis of particular proteinsmay be an important factor responsible for the reduced cellyield of the cultures in presence of high kinetin. The significanceof these observations to our understanding of the critical metaboliceffects of cytokinina is discussed. Acer pseudoplatanus cells release amino acids into their culturemedium early in the period of batch culture and largely reabsorbthem as incubation proceeds.     

Keto-acids in the Alga Chlorella

The intracellular concentration of pyruvic and -ketoglutaricacids in Chlorella when grown under various conditions has beeninvestigated. The keto-acids were assayed after quantitativeelution of their 2,4: dinitrophenylhydrazones, chromatographicallyseparated. Very small amounts of keto-acids are found in Chlorella whengrown auto-trophically. The level was much higher under heterotrophicconditions and in nitrogen starvation. Further, with nitratethe level was considerably higher than if ammonia was supplied.In the presence of glucose this differential effect occurredwith pyruvic acid, but the level of -ketoglutaric acid appearedto be stabilized. Possible interpretations of these facts arediscussed.     

The concentration of ammonia regulates nitrogen metabolism in Saccharomyces cerevisiae.

Saccharomyces cerevisiae was grown in a continuous culture at a single dilution rate with input ammonia concentrations whose effects ranged from nitrogen limitation to nitrogen excess and glucose limitation. The rate of ammonia assimilation (in millimoles per gram of cells per hour) was approximately constant. Increased extracellular ammonia concentrations are correlated with increased intracellular glutamate and glutamine concentrations, increases in levels of NAD-dependent glutamate dehydrogenase activity and its mRNA (gene GDH2), and decreases in levels of NADPH-dependent glutamate dehydrogenase activity and its mRNA (gene GDH1), as well as decreases in the levels of mRNA for the amino acid permease-encoding genes GAP1 and PUT4. The governing factor of nitrogen metabolism might be the concentration of ammonia rather than its flux.     

Fate of nitrate acquired by the tubeworm Riftia pachyptila

The hydrothermal vent tubeworm Riftia pachyptila lacks a mouth and gut and lives in association with intracellular, sulfide-oxidizing chemoautotrophic bacteria. Growth of this tubeworm requires an exogenous source of nitrogen for biosynthesis, and, as determined in previous studies, environmental ammonia and free amino acids appear to be unlikely sources of nitrogen. Nitrate, however, is present in situ (K. Johnson, J. Childress, R. Hessler, C. Sakamoto-Arnold, and C. Beehler, Deep-Sea Res. 35:1723-1744, 1988), is taken up by the host, and can be chemically reduced by the symbionts (U. Hentschel and H. Felbeck, Nature 366:338-340, 1993). Here we report that at an in situ concentration of 40 microM, nitrate is acquired by R. pachyptila at a rate of 3.54 micromol g(-1) h(-1), while elimination of nitrite and elimination of ammonia occur at much lower rates (0. 017 and 0.21 micromol g(-1) h(-1), respectively). We also observed reduction of nitrite (and accordingly nitrate) to ammonia in the trophosome tissue. When R. pachyptila tubeworms are exposed to constant in situ conditions for 60 h, there is a difference between the amount of nitrogen acquired via nitrate uptake and the amount of nitrogen lost via nitrite and ammonia elimination, which indicates that there is a nitrogen "sink." Our results demonstrate that storage of nitrate does not account for the observed stoichiometric differences in the amounts of nitrogen. Nitrate uptake was not correlated with sulfide or inorganic carbon flux, suggesting that nitrate is probably not an important oxidant in metabolism of the symbionts. Accordingly, we describe a nitrogen flux model for this association, in which the product of symbiont nitrate reduction, ammonia, is the primary source of nitrogen for the host and the symbionts and fulfills the association's nitrogen needs via incorporation of ammonia into amino acids.     

Involvement of nitrogen metabolism in the triggering of ethanol fermentation in aerobic chemostat cultures of Saccharomyces cerevisiae.

We have investigated whether central nitrogen metabolism may influence the triggering of ethanol fermentation in Saccharomyces cerevisiae strain CEN.PK122 grown in the presence of different N-sources (ammonia, glutamate, or glutamine) under conditions in which the carbon to nitrogen (C : N) ratio was varied. An exhaustive quantitative evaluation of yeast physiology and metabolic behavior through metabolic flux analysis (MFA) was undertaken. It is shown that ethanol fermentation is triggered at dilution rates, D (growth rate), significantly lower (D=0.070 and 0.074 h(-1) for glutamate and glutamine, respectively, and D=0.109 h(-1) for ammonia) under N- than C-limitation (approximately 0.18 h(-1) for all N-sources). A characteristic specific rate of glucose influx, q(Glc), for each N-source at Dc, i.e., just before the onset of respirofermentative metabolism, was determined (approximately 2.0, 1.5, and 2.5, for ammonia, glutamate, and glutamine, respectively). This q(Glc) was independent of the nutritional limitation though dependent on the nature of the N-source. The onset of fermentation occurs when this "threshold q(Glc)" is overcome. The saturation of respiratory activity appears not to be associated with the onset of fermentation since q(O(2)) continued to increase after Dc. It was remarkable that under respirofermentative conditions in C-limited chemostat cultures, the glucose consumed was almost completely fermented with biomass being synthesized from glutamate through gluconeogenesis. The results obtained show that the enzyme activities involved in central nitrogen metabolism do not appear to participate in the control of the overflow in carbon catabolism, which is driven toward ethanol production. The role of nitrogen metabolism in the onset of ethanol fermentation would rather be realized through its involvement in setting the anabolic fluxes directed to nitrogenous macromolecules. It seems that nitrogen-related anabolic fluxes would determine when the threshold glucose consumption rate is achieved after which ethanol fermentation is triggered.     

Starch Synthesis from Exogenous Sugars in Tobacco Leaf Discs

Sets of discs were taken from leaves of destarched tobacco plants(Nicotiana tabacum L. cv. xanthii) and floated on solutionsof sucrose or glucose in the dark. Abundant starch was formedin the youngest leaves but there was a marked decline with leafage.By contrast, when replicate sets of discs were floated on waterand illuminated, photosynthetic starch formation was similarin the differently aged leaves. Uptake of sugar, measured bydry weight increases and incorporation of [¹⁴C]sucrose, wasnot dependent on leaf age. The possibility that physiologicalchanges, relating to ageing and import/export status of theleaf, regulate the metabolism of sugar to starch was examined.Increasing retention of sugar in the minor veins is likely tobe a major factor. Invertase activities were measured and foundto be similar in the differently aged leaves. Respiration ratesdeclined with increasing leaf age. Speculations concerning changesin selective permeability of the chloroplast membrane are alsodiscussed.