Studies on the Nitrogen Metabolism in Ectomycorrhizae

Free and bound amino acids in the mycorrhizal and nonmycorrhizal root systems of Pinus nigra Arnold and Corylus avellna L. grown under semi-natural conditions were analyzed through automatic amino acid analyzers. Tuber brumale Vitt. and T. melanosporum Vitt. were the respective fungal symbionts. Arginine and citrulline were found to accumulate in large quantities in the free pool of the uninoculated P. nigra and C. avellana root systems respectively. In the mycorrhizal root systems these substances decreased in their levels with a parallel increase in the concentrations of glutamine and asparagine. Implications of these changes are discussed with reference to ectomycorrhizae. In general the majority of the identified free amino acids were found in larger concentrations in both the types of mycorrhizal root systems. Results obtained suggested that this may not be due to proteolysis but due to increased biosynthesis. Possible interrelationships in ectomycorrhizal root systems with reference to nitrogen metabolism are presented in the form of a scheme.     

Studies on Nitrogen Metabolism in Tobacco Plants

Δ¹-Pyrroline-5-carboxylate reductase has been considerably purified from tobacco leaves. This enzyme uses NADPH or NADH for the formation of proline, although the former is better used. This enzyme was found in washed chloroplast extract as well as in cytoplasmic fluid and utilized NADPH, formed by the photosynthetic NADP reduction, for the sythesis of proline in the light.     

Studies on Nitrogen Metabolism in Tobacco Plants A

The effect of age on non-protein constituents of tobacco leaves (N. tabacum L., var. Bright Yellow) has been studied. For this purpose leaves in three different stalk positions, upper, middle and lower, which represent young, mature and over-mature leaves, respectively, were harvested in the day-time and at night.

The total amino nitrogen content both in the day-time and at night decreases from the upper leaf position downwards. As for the individual groups, the content of both upper and middle leaves increases in the day-time and that of the lower ones increases at night.

In general, the content of the individual amino acids is high in the upper leaves and low in the lower ones. Proline and γ-aminobutyric acid, as a ratio of the total amino acid content, show a marked difference with position, in other words with age of the leaves.

The levels of proline decrease very sharply from the upper leaf position downwards and that of γ-aminobutyric acid exhibits an opposite trend in both samples at night and in the day-time. These trends are very prominent in the case of the midribs.

The contents of other amino acids, regardless of position, show similar trends with time to those reported in the previous paper₁₎, and the aspartic acid content increases at night.     

Studies on Nitrogen Metabolism in Tobacco Plants,B

Nitrate reductase from the leaves of Burley 21 tobacco (Nicotiana tabacum L.) was partially purified by ammonium sulfate fractionation, protamine sulfate treatment and calcium-phosphate gel adsorption.

The enzyme has optimum pH at 7.4 and is specific for reduced diphosphopyridine nucleotide (DPNH) as the electron donor. The nitrite formed increased in proportion to the rate at which DPNH disappeared in the reaction mixtures. Addition of flavin adenine dinucleo-tide (FAD) to the assay system enhanced the activity. FAD content in the “highly purified” enzyme was also determined. The enzyme was sensitive to heavy metals and SH-group inhibitors.

Discussions are presented on the metal and the properties of the enzyme in comparison to those published on other higher plants.     

Studies on the Nitrogen Metabolism in Tobacco Plants. A.

The nitrogenous compounds of tobacco saps have been studied both qualitatively and quantitatively and the following results were obtained.

(1) Nitrate nitrogen accounts for 40 to 70% of the total nitrogen and the rest is composed mostly of amino and alkaloid nitrogen.

(2) Amides and basic amino acids compose a large part of the amino and amide nitrogen. Among the amino acids and amides of the tobacco saps glutamine is the highest in the content and asparagine, lysine, leucine and serine follow glutamine.

(3) Topping procedure increased remarkably the alkaloid contents in the sap but decreased the amino acid nitrogen as compared with those of the untopped plant sap.     

Nitrogen Metabolism in Senescing Leaves

Leaf senescence is a highly organized process and not a passive decay. Photosynthesizing mesophyll cells lose their functions in an early phase, while the epidermal layer with the stomates and the phloem remains functional throughout senescence. The subcellular compartmentation is maintained and allows the cooperation of different organelles in the remobilization of constituents. Nitrogen metabolism changes at the onset of senescence from assimilation to remobilization. Enzymes involved in nitrate reduction are lost, while some enzymes of intermediary nitrogen metabolism are maintained longer, and some catabolic enzymes reach highest activities during senescence. Chloroplasts are dismantled early, but mitochondria remain active and may fuel remobilization processes. Chloroplast proteins are degraded, and this nitrogen fraction can be translocated via the phloem from senescing leaves to sinks within the same plant. In contrast, chlorophyll is degraded, fragments produced reach the vacuole, and catabolites accumulate there. Nuclear DNA is maintained until a very late phase. The export of nitrogen from senescing plant parts is important for the economic use of this macronutrient. The regulation of senescence at the whole plant level as well as at the molecular level is only rudimentarily known, although interesting new aspects have been presented recently.     

Nitrogen Metabolism in Paramecium aurelia

Nitrogen in cell fractions of Paramecium aurelia varied according to the growth medium. Trichloroacetic acid-soluble fractions of cells were chromatographer. Adenine, adenosine, guanine, guanosine, hypoxanthine, aspartic acid, glutamic acid, histidine, lysine, proline, and phenylalanine were identified. Fyrimidines and xanthine, or their respective ribosides and ribotides, were not detected. Ammonia was released into the medium by both actively growing and "resting" cells. Culture fluids of "resting"cells also contained hypoxanthine and lesser amounts of adenine and guanine. Urea, uric acid, creatine, cretonne, and ailantoin were absent.
Pyrimidine nitrogen seems excreted as dihydrouracil. The following enzymes were detected in homogenates and cell-free preparations: nucleotidases, nucleoside hydrolases, and cytidine deaminase. Urease, uricase, adenase, guanase, xanthine oxidase, adenosine deaminase, and 5'-adenylic acid deaminase were not present in this organism.
Purine and pyrimidine incorporation into nucleic acids was investigated by the use of radioactive tracers. Guanosine gives rise to nucleic-acid guanine and adenine; adenosine was precursor to nucleic acid adenine only. Formate was incorporated into purines; glycine was not. P. aurelia can interconvert cytidine and uridine; both give rise to nucleic acid thymine. The methyl group of thymine may be derived from formate.     

Studies on the Metabolism of Aspergilli

Studies were made on the effect of water level of culture medium on the mycelial compositions and enzyme production in Aspergillus sojae K. S. The mold was grown on the media of various water levels made of powder of defatted soybean and wheat granule. The mycelia grown on the medium of low water level produced more protease and α-amylase, consumed more oxygen, formed less ammonia, and were richer in 2 n H₂SO₄-soluble glycogen, 60% H₂SO₄-soluble carbohydrates, protein and RNA per mg dry weight than the mycelia grown on the medium of high water level. Chromatographic analyses were carried out for nucleotides, sugar phosphates and free carbohydrates in cold TCA-soluble fraction of the mycelia.     

Studies on the Metabolism of Aspergilli

This report is concerned with the time course of changes of several enzymes during endogenous respiration. It was observed that enzymes such as deaminases, amidases and transaminases found in the mycelia increased in activity more or less during endogenous respiration. It was assumed that enzyme formation occurred as the result of glucose starvation or depression of carbohydrate metabolism during endogenous respiration of the mold mycelia on buffer solution.     

Studies on the Metabolism of Aspergilli

Studies were made on the endogenous respiration of Aspergillus sojae K.S. Observing the changes of Kjeldahl-nitrogen in each fraction of the mycelial components, the author concluded that pool amino acids, bound amino acids, protein, nucleic acids and nucleotides covered whole of the nitrogenous reserves available for endogenous respiration in the mycelia. A study was carried out on the effect of preincubation with glucose or amino acids on endogenous respiration. Stimulation of either oxygen uptake, protein breakdown or ammonia formation was observed during respiration of the mycelia incubated with a suitable concentration of azide, 2,4-dinitrophenol, potossium fluoride, monoiodoacetic acid or ethylenediaminetetraacetic acid. Ammonia formation accompanied with endogenous respiration seemed to proceed inversely by the influence of energy yielding reaction.     

Studies on the Metabolism of Aspergillii

Endogenous respiration of Aspergillus sojae K.S. was studied in terms of biochemical analysis. It was found that the different kind of substrates was utilized for the endogenous respiratoin according to C:N ratio of the agar medium on which the mold was grown. In the mycelial mats grown on the medium of low C:N value, pool amino acids, protein, and nucleic acids were mainly utilized from the beginning while carbohydrate or lipid displayed a minor role. The corresponding amount of ammonia was formed. On the other hand, in the mycelial mats grown on the medium of rather high C:N value, carbohydrate or lipid was the major substrate of endogenous respiration in the early stages of incubation. The utilization of the nitrogenous materials and the accompanying formation of ammonia got to start only after the lapse of several hours of incubation.