The Assimilation of Nitrogen from Ammonium Salts and Nitrate by Fungi

1. The assimilation of inorganic nitrogen by Scopulariopsis brevicaulisand some physiologically similar species has been studied. Theirfailure to assimilate completely from ammonium sulphate hasbeen shown to be due to the fall in pH of the medium inducedby the initial uptake of ammonia.
2. Complete assimilation ofammonia takes place in the presenceof the neutral salts ofeach of thirteen organic acids investigated.The organic acidsact primarily through their buffering effectwhich preventsor slows down the fall in pH. They are not specificallyrequiredfor ammonia assimilation by these fungi and can beeffectivelyreplaced by certain inorganic buffers.
3. The influence of severalexternal factors on the rate of assimilationof ammonia, nitrate,and nitrite has been studied in S. brevicaulis.In correspondingconditions the mycelium assimilates ammoniamore rapidly thannitrate over a wide range of conditions.
4. Ammonia, even invery low concentration, completely suppressesnitrate assimilationwhen both sources of nitrogen are presenttogether. Nitrite,however, is assimilated simultaneously withammonia. It is thereforeconcluded that ammonia blocks the reductionof nitrate to nitriteby the fungus.
5. The suppression of nitrate assimilation inthe presence of ammoniais common to many mould fungi besidesS. brevicaulis, and isbelieved to have adaptive significancein natural habitats.
6. The nitrate-reducing and assimilatingsystem is formed, evenwhen S. brevicaulis is grown in completeabsence of nitrate(ammonia medium with organic acid). It comesinto action rapidlywhen the inhibiting effect of ammonia isremoved. Similarly,nitrate-grown mycelium is capable of assimilatingammonia atmaximal rate without any adaptive lag.

     

Preferential Assimilation of Ammonium Ions from Ammonium Nitrate Solutions by Apple Seedlings

Apple seedlings, Pyrus malus L., were grown in complete nutrient solutions containing nitrate, ammonium, or ammonium plus nitrate as the nitrogen source. Uptake of nitrogen was calculated from depletion measurements of the nutrient solutions and by using ¹⁵N labelled nitrate and ammonium salts. If the plants received nitrogen as ammonium only or as nitrate only, the amounts of nitrogen taken up were similar. However, if the seedlings were supplied with ammonium nitrate, the amount of nitrate-nitrogen assimilated was only half that of ammonium. Nevertheless, if ammonium and nitrate were supplied to a plant with a split-root system, with each root half receiving a different ion, the uptakes were similar. The possibility of independent inhibition by ammonium of both nitrate uptake and reduction in the roots is discussed.     

Autotrophic Carbon Dioxide Assimilation in Thermoproteales Revisited

For Crenarchaea, two new autotrophic carbon fixation cycles were recently described. Sulfolobales use the 3-hydroxypropionate/4-hydroxybutyrate cycle, with acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the carboxylating enzyme. Ignicoccus hospitalis (Desulfurococcales) uses the dicarboxylate/4-hydroxybutyrate cycle, with pyruvate synthase and phosphoenolpyruvate carboxylase being responsible for CO₂ fixation. In the two cycles, acetyl-CoA and two inorganic carbons are transformed to succinyl-CoA by different routes, whereas the regeneration of acetyl-CoA from succinyl-CoA proceeds via the same route. Thermoproteales would be an exception to this unifying concept, since for Thermoproteus neutrophilus, the reductive citric acid cycle was proposed as a carbon fixation mechanism. Here, evidence is presented for the operation of the dicarboxylate/4-hydroxybutyrate cycle in this archaeon. All required enzyme activities were detected in large amounts. The key enzymes of the cycle were strongly upregulated under autotrophic growth conditions, indicating their involvement in autotrophic CO₂ fixation. The corresponding genes were identified in the genome. ¹⁴C-labeled 4-hydroxybutyrate was incorporated into the central building blocks in accordance with the key position of this compound in the cycle. Moreover, the results of previous ¹³C-labeling studies, which could be reconciled with a reductive citric acid cycle only when some assumptions were made, were perfectly in line with the new proposal. We conclude that the dicarboxylate/4-hydroxybutyrate cycle is operating in CO₂ fixation in the strict anaerobic Thermoproteales as well as in Desulfurococcales.Two new autotrophic carbon fixation cycles have recently been discovered in the Crenarchaea, one of the two subgroups of the Archaea. The 3-hydroxypropionate/4-hydroxybutyrate cycle functions in the aerobic autotrophic Sulfolobales (7) and the dicarboxylate/4-hydroxybutyrate cycle (Fig. ​(Fig.1)1) in the anaerobic autotrophic Ignicoccus hospitalis, belonging to the Desulfurococcales (27). These pathways have in common the synthesis of succinyl-coenzyme A (CoA) from acetyl-CoA and two inorganic carbons, although this is accomplished in quite different ways and using different carboxylases. In the 3-hydroxypropionate/4-hydroxybutyrate cycle, acetyl-CoA/propionyl-CoA carboxylase fixes two molecules of bicarbonate, and in the dicarboxylate/4-hydroxybutyrate cycle, pyruvate synthase and phosphoenolpyruvate (PEP) carboxylase are the two carboxylating enzymes. Yet, the regenerations of acetyl-CoA, the primary CO₂ acceptor, from succinyl-CoA are similar in the two pathways.Open in a separate windowFIG. 1.Dicarboxylate/4-hydroxybutyrate cycle for autotrophic CO₂ fixation, as proposed for T. neutrophilus. Enzymes: 1, pyruvate synthase (reduced MV); 2, pyruvate-water dikinase; 3, PEP carboxylase; 4, malate dehydrogenase (NADH); 5, fumarate hydratase; 6, fumarate reductase (reduced MV); 7, succinyl-CoA synthetase (ADP forming); 8, succinyl-CoA reductase (NADPH); 9, succinic semialdehyde reductase (NADPH); 10, 4-hydroxybutyrate-CoA ligase (AMP forming); 11, 4-hydroxybutyryl-CoA dehydratase; 12, crotonyl-CoA hydratase; 13, (S)-3-hydroxybutyryl-CoA dehydrogenase (NAD⁺); 14, acetoacetyl-CoA β-ketothiolase. Fd_red, reduced ferredoxin.Acetyl-CoA regeneration is as follows. The CO₂ fixation product succinyl-CoA is reduced to 4-hydroxybutyrate, which is activated to 4-hydroxybutyryl-CoA and then dehydrated to crotonyl-CoA by 4-hydroxybutyryl-CoA dehydratase. This radical [4Fe-4S] and flavin adenine dinucleotide-containing dehydratase (11, 37) is considered a key enzyme of the 4-hydroxybutyrate part of each pathway. Its product, crotonyl-CoA, is further converted to acetoacetyl-CoA and then to two acetyl-CoA molecules, closing the cycle and generating an additional molecule of acetyl-CoA for biosynthesis. Therefore, two different autotrophic pathways in different crenarchaeal orders share many common enzymes and intermediates.In this context, the order Thermoproteales would constitute an exception within the Crenarchaea, since the reductive citric acid cycle was proposed for Thermoproteus neutrophilus (6, 48-50, 55) and Pyrobaculum islandicum (26). T. neutrophilus is a strictly anaerobic hyperthermophilic archaeon growing autotrophically by reducing sulfur with hydrogen at 85°C and neutral pH (19). It can also assimilate organic compounds, such as acetate or succinate, but only in the presence of CO₂ and H₂, i.e., in a mixotrophic way (48).In the reductive citric acid cycle, succinyl-CoA is further transformed with 2 CO₂ to citrate, followed by citrate cleavage to oxaloacetate and acetyl-CoA. This requires two characteristic enzymes, 2-oxoglutarate synthase (2-oxoglutarate-ferredoxin oxidoreductase) and ATP citrate lyase. The proposal of the functioning of the reductive citric acid cycle in T. neutrophilus was based on the results of a ¹³C retrobiosynthetic analysis of the central carbon metabolism, using ¹³C-labeled succinate and acetate as an additional carbon source, following its incorporation into cellular building blocks. The ¹³C enrichment data of, e.g., glutamate, which is directly derived from 2-oxoglutarate, were consistent with the operation of a reductive citric acid cycle only when further assumptions were made (55). The activities of the enzymes of this cycle were demonstrated with extracts of autotrophically grown cells. However, the measured 2-oxoglutarate synthase and ATP-citrate lyase activity levels were very low and could not support the reported growth rate under autotrophic conditions (6, 48).The recent sequencing of the genome of Pyrobaculum aerophilum, belonging to the Thermoproteales (20), revealed a surprising feature, the presence of a 4-hydroxybutyryl-CoA dehydratase gene without the presence of an ATP-citrate lyase gene. Similar gene patterns are found in the genomes of T. neutrophilus as well as Pyrobaculum calidifontis and P. islandicum, sequenced by the DOE Joint Genome Institute (http://www.jgi.doe.gov/). This indicates a possible functioning of the dicarboxylate/4-hydroxybutyrate cycle in Thermoproteales and brings into question the involvement of the reductive citric acid cycle in autotrophic CO₂ fixation. This study has reinvestigated the pathway of autotrophic CO₂ fixation in Thermoproteus neutrophilus. We provide different lines of evidence for the operation of the dicarboxylate/4-hydroxybutyrate cycle.     

Effect of Glucose on Carbon Dioxide Assimilation and Substrate Oxidation by Ferrobacillus ferrooxidans

Ferrobacillus ferrooxidans, grown on either elemental sulfur or ferrous sulfate, was able to use either substrate as an energy source for the assimilation of CO(2). In both cases, 0.01 mumole of carbon was incorporated per mumole of oxygen utilized. Glucose inhibited substrate oxidation and CO(2) fixation. Sulfur and iron oxidation were inhibited 5 to 15% and 40 to 50%, respectively, in the presence of 10% glucose. Under the same conditions, CO(2) assimilation was inhibited 50% with elemental sulfur as the energy source, and was almost totally inhibited when ferrous iron was used.     

Nitrogen Fixation and Carbon Dioxide Assimilation in Rhizobium japonicum

In free-living Rhizobium japonicum cultures, the stimulatory effect of CO₂ on nitrogenase (acetylene reduction) activity was mediated through ribulose bisphosphate carboxylase activity. Two mutant strains (CJ5 and CJ6) of R. japonicum defective in CO₂ fixation were isolated by mitomycin C treatment. No ribulose bisphosphate carboxylase activity could be detected in strain CJ6, but a low level of enzyme activity was present in strain CJ5. Mutant strain CJ5 also exhibited pleiotropic effects on carbon metabolism. The mutant strains possessed reduced levels of hydrogen uptake, formate dehydrogenase, and phosphoribulokinase activities, which indicated a regulatory relationship between these enzymes. The CO₂-dependent stimulation of nitrogenase activity was not observed in the mutant strains. Both mutant strains nodulated soybean plants and fixed nitrogen at rates comparable to that of the wild-type strain.     

Promotion of Assimilation of Ammonium Ions by Simultaneous Application of Nitrate and Ammonium Ions in Radish Plants

When radish plants were grown in nutrient solutions that containedammonium ions (NH₄⁺) as the sole source of nitrogen, they grewpoorly and accumulated high levels of NH₄⁺ in their leaves.However, radish plants cultured in 5 mM NH₄⁺ plus 1 mM NO₃^–(a ratio of 5 : 1 in forms of nitrogen; referred to as 5:lmix-N)grew well and accumulated very low levels of NH₄⁺ in their leaves.After radish plants were cultured in solutions that containedNO₃^–, or NH₄⁺, or 5: lmix-N for a week, they were thensupplied with the same nitrogen source labeled with ¹⁵N forone day. The uptake of ¹⁵N from labeled NH₄⁺ into total nitrogenwas the highest in plants supplied with 5:1mix-N. These plantsconverted far fewer labeled NH₄⁺ into free NH₄⁺ than did NH₄⁺-fedplants, but converted many more labeled NH₄⁺ into the insolublefraction than did NH₄⁺- or NO₃^–-fed plants. The presence of a small amount of nitrate was shown to stimulatethe assimilation of ammonium ions and the synthesis of proteins. (Received October 26, 1988; Accepted January 24, 1989)     

The Response of Nitrate Reductase Activity and Nitrate Assimilation in Maize Roots to Growth Regulators at Acidic pH

Nitrate and total nitrogen contents, and nitrate reductase (NR) activity of the excised maize roots in buffered or unbuffered nitrate solution (at pH 6.5 or 4.5) as affected by putrescine (PUT), abscisic acid (ABA) and salicylic acid (SA) were investigated. In unbufferred solution, the NR activity was lower at pH 4.5 as compared to that at pH 6.5, but in bufferred solution the activity was higher at lower pH. Supply of 100 µM PUT or 500 µM SA, promoted NR activity and 50 µM ABA inhibited the activity at pH 6.5. However, at pH 4.5, PUT and SA inhibited NR activity and ABA had no effect. In most cases, the increase in NR activity was positively correlated with total organic nitrogen and a negatively with nitrate content. A reverse situation was found when NR activity was inhibited by the growth regulators.     

Isotope Fractionation in Photosynthetic Bacteria during Carbon Dioxide Assimilation

The δ _PDB¹³C values have been determined for the cellular constituents and metabolic intermediates of autotrophically grown Chromatium vinosum. The isotopic composition of the HCO₃^- in the medium and the carbon isotopic composition of the bacterial cells change with the growth of the culture. The δ _PDB¹³C value of the HCO₃^- in the media changes from an initial value of −6.6‰ to +8.1‰ after 10 days of bacterial growth and the δ _PDB¹³C value of the bacterial cells change from −37.5‰ to −29.2‰ in the same period. The amount of carbon isotope fractionation during the synthesis of hexoses by the photoassimilation of CO₂ has a range of −15.5‰ at time zero to −22.0‰ after 10 days. This range of fractionation compares to the range of carbon isotope fractionation for the synthesis of sugars from CO₂ by ribulose 1,5-diphosphate carboxylase and the Calvin cycle.     

粪产碱菌（Alcaligenes faecalis）吸收氢和同化CO2的特性

应用同位素氚(T_2)和~13C(~13CO_2),证明了水稻联合固氮菌——粪产碱菌A—15是一种含有吸氢酶的兼性化能自养细菌,具有较强的吸氢能力,吸氨酶活性可达到13.11μmol H_2 ml~(-1) cultureh~(-1);同时,它还可利用H_2为能源同化CO_2营化能自养生活,其RuBPC活性为24.65 nmolCO_2 mg~(-1) protein min~(-1)。无论在自养还是异养条件下,H_2都支持、并促进固氮活性。粪产碱菌培养在N_2条件下比在NH_4~ 条件下能积累更多的多聚-β羟基丁酸(PHB)。     

Carbon Dioxide Compensation Points in Maize and Pelargonium

The carbon dioxide compensation paint established by maize leaves in 10,000 lux at 25°C was less Than 2 μl/l. whereas that achieved by Pelargonium under the same conditions was greater than 60 μl/l. Maize and Pelargonium leaves together in the same leaf chamber established a compensation point between 9 and 19 μl/l. It is concluded that it is a difference in the efficiency of absorption of CO₂ which leads to the marked difference between maize and Pelargonium, rather than the absence of photorespiration in maize or anatomicad differences between the leaves of the two species.     

Nitrate and Ammonium Assimilation by Roots of Maize (Zea mays L.) Seedlings as Investigated by In Vivo 15N-NMR

¹⁵N-Nuclear Magnetic Resonance (NMR) was used to study nitrogenassimilation in apices of maize roots in vivo, perfused eitherwith ¹⁵NO     

Inhibition of Nitrate Assimilation in Roots in the Presence of Ammonium: The Moderating Influence of Potassium

Experiments were conducted to investigate the effect of concentrationof NH₄⁺ in nutrient solution on root assimilation of NO₃^–and to determine whether the NH₄⁺NO₃^– interaction wasmodified in the presence of K⁺. Dark-grown, detopped corn seedlings(cv. Pioneer 3369A) were exposed for 8 h to 0.15 mM Ca(NO₃)₂and varying concentrations of (NH₄)₂SO₄ in the absence or presenceof 0.15 mM K₂SO₄. The accelerated phase of NO₃^– uptakeappeared most sensitive to restriction by additions of 0.15mM (NH₄)₂SO₄. In the absence of K⁺, the restriction increasedonly slightly even when solution (NH₄)₂SO₄, was increased from0.15 mM to 12.5 mM which was accompanied by an increase of NH₄⁺in the tissue from about 7.0 to 35 µmol g^–1 fr.wt. of root. Increasing concentrations of solution NH₄⁺ progressivelyinhibited net K+ uptake. At the highest solution NH₄⁺ concentrations,there was an initial net efflux of K⁺ and no net influx occurredduring the treatment period. The severity of the NH₄)SO₄ restrictionof NO₃^– uptake was moderated considerably in the presenceof K⁺ as long as a net influx of K⁺ occurred. However, net influxof K⁺ was not associated with alteration of NH₄⁺ uptake, assimilation,or accumulation in the root tissue. The lack of correlationbetween the severity of restriction of NO₃^– uptake andendogenous NHJ suggested the restriction resulted from an effectexerted by exogenous NH₄⁺ which tended to saturate at lowersolution NHJ concentrations or by inhibitory factors generatedduring assimilation of NH₄⁺. Several mechanisms were postulatedto account for the moderating influence of K⁺. In all experiments,root NO₃^– reduction was restricted by the presence ofambient NH₄⁺. The quantitative decreases in reduction tendedto be less than decreases in NO₃^– uptake and therefore,could result from inhibition solely of uptake with subsequentlimitation in availability of substrate for the reduction process,but the possibility of a direct effect on reduction could notbe excluded.     

Role of Potassium in Carbon Dioxide Assimilation in Medicago sativa L

Alfalfa was grown hydroponically in 0, 0.6, and 4.8 millimolar K in order to determine the influence of tissue level of K on photosynthesis, dark respiration, photorespiration, stomatal and mesophyll resistance to CO₂, photosystem I and II activity, and synthesis and activity of ribulose 1,5-bisphosphate carboxylase (RuBPc).     

Nitrate Uptake and Assimilation following Nitrate Deprivation

Upon first exposure to , the uptake and reduction capacities of dark-grown corn (Zea maysL.) roots are initially low, but increase markedly within 6h. The development of the accelerated uptake rate appears to be substrate ‘induced’ as is reductase (NR), the first enzyme in the assimilatory pathway. However, the ‘induction’of uptake is independent of NR induction. The effect of deprivation was studied to determine the role of endogenous on subsequent uptake and reduction. Corn roots were ‘induced’ for 24 h in 0–5 mol m^–3 nutrient solution and then exposed for 0 to 32 h to -free nutrient solution. Uptake and reduction of were determined periodically by exposing sets of roots to a1 h pulse of 0.5 mol m^–3 . Neither uptake (4.57 µmol root^–1 h^–1)nor the percentage of absorbed reduced (27%) was changed significantly (P 0.05) by exogenous deprivation. However, the estimated ‘induced’ componentof uptake decreased significantly (50% after 32 h). Concurrently, the ‘non-induced’ basal componentof uptake increased. Previously accumulated decreased from 23 to 4.5 µmol root^–1 after 32 h of exogenous deprivation. Nearly equivalent quantities of endogenous were used for translocation and reduction during deprivation. During each 1 h pulse, the amounts of translocation and net efflux of to the uptake solution were similar. Net efflux of was strongly correlated (r = 0.991) to the amount of endogenous . The remaining endogenous and its rate of utilization were apparently sufficient to minimize a rapid declineor complete loss in both the ‘induced’ uptake state and the rate of in vivo assimilation. Key words: reduction, translocation, efflux, root, Zea mays L     

Carbon Assimilation at Low Carbon Dioxide Levels: I. APPARATUS AND TECHNIQUE

The construction and operation of a versatile apparatus forthe measurement of CO₂ exchange of detached plant parts is described. CO₂ concenteration was measured with an accuracy of about ±3per cent using a commercial infra-red gas analyser; measurementswere made at ambient CO₂ levels between 10 and 10,000 p.p.m.(0.001 per cent. and 1.0 per cent. by volume), at leaf temperaturesbetween 5°C. and 40°C. (±0.1°C.) and at lightintensities up to 2,000 foot candles. The measurements were made on either a fixed volume of gas repeatedlypassed over the leaf, or on a stream of gas passing over theleaf once only, or with any desired combination of these two. Rates of gas flow (up to 801./hr.) could be controlled to finelimits independent of changes in flow resistance and measuredwith an accuracy of at least ±1 per cent., if required.     

Influence of Nitrate and Ammonium Nutrition on the Uptake, Assimilation, and Distribution of Nutrients in Ricinus communis

Ricinus communis L. plants were grown in nutrient solutions in which N was supplied as NO₃⁻ or NH₄⁺, the solutions being maintained at pH 5.5. In NO₃⁻-fed plants excess nutrient anion over cation uptake was equivalent to net OH⁻ efflux, and the total charge from NO₃⁻ and SO₄²⁻ reduction equated to the sum of organic anion accumulation plus net OH⁻ efflux. In NH₄⁺-fed plants a large H⁺ efflux was recorded in close agreement with excess cation over anion uptake. This H⁺ efflux equated to the sum of net cation (NH₄⁺ minus SO₄²⁻) assimilation plus organic anion accumulation. In vivo nitrate reductase assays revealed that the roots may have the capacity to reduce just under half of the total NO₃⁻ that is taken up and reduced in NO₃⁻-fed plants. Organic anion concentration in these plants was much higher in the shoots than in the roots. In NH₄⁺-fed plants absorbed NH₄⁺ was almost exclusively assimilated in the roots. These plants were considerably lower in organic anions than NO₃⁻-fed plants, but had equal concentrations in shoots and roots. Xylem and phloem saps were collected from plants exposed to both N sources and analyzed for all major contributing ionic and nitrogenous compounds. The results obtained were used to assist in interpreting the ion uptake, assimilation, and accumulation data in terms of shoot/root pH regulation and cycling of nutrients.     

Regulation of Nitrate Assimilation and Nitrate Respiration in Aerobacter aerogenes

The influence of growth conditions on assimilatory and respiratory nitrate reduction in Aerobacter aerogenes was studied. The level of nitrate reductase activity in cells, growing in minimal medium with nitrate as the sole nitrogen source, was much lower under aerobic than anaerobic conditions. Further, the enzyme of the aerobic cultures was very sensitive to sonic disintegration, as distinct from the enzyme of anaerobic cultures. When a culture of A. aerogenes was shifted from anaerobic growth in minimal medium with nitrate and NH(4) (+) to aerobiosis in the same medium, but without NH(4) (+), the production of nitrite stopped instantaneously and the total activity of nitrate reductase decreased sharply. Moreover, there was a lag in growth of about 3 hr after such a shift. After resumption of growth, the total enzymatic activity increased again slowly and simultaneously became gradually sensitive to sonic disintegration. These findings show that oxygen inactivates the anaerobic nitrate reductase and represses its further formation; only after a de novo synthesis of nitrate reductase with an assimilatory function will growth be resumed. The enzyme in aerobic cultures was not significantly inactivated by air, only by pure oxygen. The formation of the assimilatory enzyme complex was repressed, however, by NH(4) (+), under both aerobic and anaerobic conditions. The results indicate that the formation of the assimilatory enzyme complex and that of the respiratory enzyme complex are regulated differently. We suggest that both complexes have a different composition, but that the nitrate reductase in both cases is the same protein.     

Translocation of 14Carbon in Tobacco following Assimilation of 14Carbon Dioxide by a Single Leaf

¹⁴CO₂ was assimilated by single leaves (presentation leaves)of tobacco plants for periods of 2–3 hours. The plantswere then kept in air in continuous light and the redistributionof radioactivity determined at various times up to 96 hours.There was a complete turnover of sucrose in the presentationleaf in about 24 hours without change in amount. Starch turnedover more slowly and simultaneously increased in amount. 20–30per cent. of the radioactivity appeared to be irreversibly incorporatedinto the presentation leaf. Of the material exported from thepresentation leaf some 3 per cent. reached the upper leavesand stem apex. Import into leaves above the presentation leafwas completed in about 6 hours. No activity appeared in leavesbelow the presentation leaf, therefore the balance of the exportedactivity was retained in the stem and roots. The distribution of radioactivity in the leaves followed a well-definedpattern determined by the vascular interconnexions. Radioautographs of stem sections provided some information concerningdistribution of radioactivity in the stem.