The nature of the organic nitrogen of soils

Summary Examination of the 6N HC1 hydrolysates from 14 different proteins indicated that a considerable proportion of the total protein nitrogen in the hydrolysates, as determined by the micro-Kjeldahl method, was not accounted for by the NH₄-N and the α amino nitrogen found in the hydrolysates. It seems clear that this hydrolysable unidentified nitrogen (HUN) originates mainly from non-amino nitrogen atoms present in arginine, tryptophan, lysine and proline. These nitrogen atoms do not satisfy the conditions necessary for reaction with ninhydrin. The amounts of each amino acid in a particular protein determine the HUN value which will be obtained for 6N HC1 hydrolysates of that protein. There is good agreement between the HUN values for a wide range of proteins when calculated from the amino acid composition of the protein and when determined experimentally. It is concluded that these findings indicate a considerably higher content of amino acid nitrogen in the organic nitrogen of soils and leaf litter than was previously considered to be the case. It is suggested that the findings support the contention that the organic nitrogen of soils contains leaf protein complexes.     

Studies on symbiotic nitrogen fixation by a new strain of tetraploid red clover (UO36)

Summary Some characteristics of the symbiotic nitrogen fixation by tetraploid red clover (UO36) have been studied in laboratory experiments. Cross-inoculations were made on tetraploid and diploid clover plants usingRhizobium isolates from both types of clover. Nodulation activity and nitrogen fixation were determined. No sign of specific strains necessary for optimal fixation of nitrogen by theUO36-symbiosis was observed in comparison to the diploid clover strain.Rhizobium cultures effective in connection with cultivation of diploid clover will probably also be effective when cultivating tetraploid clover strains.     

Variations in the amino acid composition of cyanophycin in the cyanobacterium Synechocystis sp. PCC 6308 as a function of growth conditions

Gas chromatography-mass spectrometry studies of the nitrogen isotopic composition of the N-trifluoroacetyl n-butyl ester derivatives of the amino acids from isolated hydrolyzed cyanophycin from ¹⁵N-enriched cells led to two major findings: (1) the amino acid composition of this granular polypeptide, isolated using procedures optimized for extracting and purifying cyanophycin from cells in the stationary growth phase, varied with the culture growth condition; (2) the rate of incorporation of exogenous nitrate differed for each nitrogen atom of the amino acid constituents of cyanophycin or cyanophycin-like polypeptide. Arginine and aspartic acid were the principle components of cyanophycin isolated from exponentially growing cells and from light-limited stationary phase cells, with glutamic acid as an additional minor component. The cyanophycin-like polypeptide from nitrogen-limited cells contained only aspartic and glutamic acids, but no arginine. The glutamic acid content decreased and arginine content increased as nitrate was provided to nitrogen-limited cells. These cells rapidly incorporated nitrate at different rates at each cyanophycin nitrogen site: guanidino nitrogens of arginine>aspartic acid >-amino nitrogen of arginine>glutamic acid. Little media-derived nitrogen was incorporated into cyanophycin of exponentially growing cells during one cellular doubling time.Abbreviations asp-TAB, glu-TAB, arg-TAB N-Trifluoroacetyl n-butyl ester derivatives of aspartic acid, glutamic acid and arginine, respectively - CAP chloramphenicol - CF correction factor - TFAA Trifluoroacetic anhydride - MBTFA N-Methyl-bis-trifluoroacetamide     

不同形态氮素施肥对小黑杨幼苗生长的影响

以小黑杨当年播种苗为材料,研究了不同施氮量（12,24和48 mg·株^-1）和不同形态氮素（有机氮和无机氮）施肥对小黑杨幼苗生长的影响,以探讨小黑杨对氮基酸类有机氮素施肥的生长响应。结果表明：无论施无机氮（硝酸铵）还是有机氮（精氨酸）,小黑杨幼苗的苗高、地径、总生物量都是随施氮量的增加而增加,但是中等施氮量处理的幼苗氮利用效率最高。无机氮（硝酸铵）和有机氮（精氨酸）处理的幼苗在相同施氮水平下生长表现无明显差异,施用有机氮可以与施用无机氮获得相同的促进苗木生长的效果。不同氨基酸及其组合肥料施用对小黑杨幼苗生长的影响显著。单一氨基酸施肥情况下,施用精氨酸促进苗木生长的效果最好,谷氨酸次之,甘氨酸最差;氨基酸组合施肥情况下,有精氨酸的组合施肥苗木生长好,有甘氨酸的组合施肥苗木生长差。不同氨基酸施肥处理对小黑杨幼苗各器官氮含量没有明显影响。     

Root exudates: a pathway for short-term N transfer from clover and ryegrass

The short-term transfer of nitrogen (N) from legumes to grasses was investigated in two laboratory studies. One study was done in pots where the roots of white clover (Trifolium repens L.) and perennial ryegrass (Lolium perenne L.) were allowed to co-exist, and a second study was performed using a micro-lysimeter system designed to maintain nutrient flow from the clover to the grass, whilst removing direct contact between the root systems. The ¹⁵N-dilution technique was used to quantify the transfer of N between species. Levels of ammonia and amino acids were measured in root exudates. The amounts of N transferred were in the same order of magnitude in both the pot and micro-lysimeter experiments. In the micro-lysimeter experiment, 0.076 mg of N were transferred per plant from clover to ryegrass during the course of the experiment. Ammonium exudation was much higher than amino acid exudation. The most abundant amino acids in both clover and ryegrass root exudates were serine and glycine. However, there was no correlation between the free amino acid profile of root extracts and exudates for both plant species: Asparagine was the major amino acid in clover roots, while glutamine, glutamate and aspartate were the major amino acids in ryegrass roots. Comparison of exudates obtained from plants grown in non-sterile or axenic conditions provides evidence of plant origin of ammonium, serine and glycine.     

Chemical Properties of Syringomycin and Syringotoxin: Toxigenic Peptides Produced by Pseudomonas syringae

Syringomycin (SR) and syringotoxin (ST), wide spectrum antibiotics and phytotoxins isolated from ecotypic strains of Pseudomonas syringa , were purified to homogeneity and compared for their physicochemical properties. Acid hydrolysates of SR and ST were analysed for ninhydrin-reactive components by paper chromatography and the Durrum single-column method of amino acid analysis. Both active and base inactivated preparations of SR yielded substances tentatively identified as serine, phenylalanine, an unidentified basic amino acid, and arginine in a 2:1:2:1 mole ratio, respectively. Preparations of SR from ecotypic strains of P. syringae from pear, peach and millet hosts, had an identical amino acid composition which appeared to exclude a potential role of SR in the plant host specificity of P. syringae . ST isolated from a strain of P. syringae from a citrus host, contained substances tentatively identified as threonine, serine, glycine, ornithine, and the same unidentified basic amino acid found in SR in a 1:1:1:1:1 mole ratio. Although autoradiographs of paper chromatograms of acid hydrolysates of ¹⁴C-SR and various chromogenic reagents did not indicate the presence of substances other than amino acids, the nitrogen content of SR by combustion analyses was lower than expected which suggested the possible presence of another component.     

Effect of Ploidy and Flowering Type of Red Clover Cultivars and of Isolate Origin on Severity of Clover Rot, Sclerotinia trifoliorum

Two complementary experiments were conducted in a controlled environment to elucidate the interactions between the fungus Sclerotinia trifoliorum Erikss. and red clover (Trifolium pratense L.). In one of these studies, two hardened diploid red clover cultivars (cvs) were inoculated with 20 isolates of S. trifoliorum of various geographic origins. In the other study, 20 red clover cvs, diploid or tetraploid, including late and medium‐late flowering types, were inoculated with two isolates of the fungus. Prior to inoculation, some plants were hardened by subjecting them to a low temperature and light treatment mimicking autumn conditions. Late flowering cvs were found more resistant than medium‐late ones. Isolates collected in the northern region, where late cvs are grown, were significantly more aggressive than isolates from southern locations, where medium‐late cvs are more prevalent. Such an adaptation has not previously been reported for this fungus. This is the first report concerning flowering type and resistance in red clover. Tetraploids were generally not more resistant than diploids. A hardening procedure for red clover plants was found to be a prerequisite for detecting the differences in disease resistance.     

Effects of combined nitrogen on anapleurotic carbon assimilation and bleeding sap composition in Phaseolus vulgaris L.

Dwarf french beans, Phaseolus vulgaris L., were grown with or without inoculation with rhizobia (strain 3644), and with or without a combined nitrogen source (nitrate or ammonium ions). The distribution of radioactivity into products of dark ¹⁴CO₂ assimilation was studied in roots or nodules from these plants. A detailed study was also made of the distribution and rates of excretion of nitrogen in xylem bleeding sap in 28 day old plants grown on the various sources of nitrogen. Whereas detached nodules accumulated radioactive glycine, serine and glutamate when incubated with ¹⁴CO₂, bleeding sap from plants root fed ¹⁴CO₂ contained low levels of radioactivity in these compounds but higher levels in allantoin. Chemical analysis showed allantoin to be the major compound transported in the xylem of nodulated plants, whether or not they were fed on combined nitrogen. In contrast uninoculated plants accumulated mainly amino acids in the bleeding sap, the amount and chemical composition of which depended on the combined nitrogen source.Abbreviations PEP phosphoenol pyruvate - OAA oxaloacetate     

Nitrogen Nutrition and Xylem Sap Composition of Peanut (Arachis hypogaea L. cv Virginia Bunch)

The principal forms of amino nitrogen transported in xylem were studied in nodulated and non-nodulated peanut (Arachis hypogaea L.). In symbiotic plants, asparagine and the nonprotein amino acid, 4-methyleneglutamine, were identified as the major components of xylem exudate collected from root systems decapitated below the lowest nodule or above the nodulated zone. Sap bleeding from detached nodules carried 80% of its nitrogen as asparagine and less than 1% as 4-methyleneglutamine. Pulse-feeding nodulated roots with ¹⁵N₂ gas showed asparagine to be the principal nitrogen product exported from N₂-fixing nodules. Maintaining root systems in an N₂-deficient (argon:oxygen, 80:20, v/v) atmosphere for 3 days greatly depleted asparagine levels in nodules. 4-Methyleneglutamine represented 73% of the total amino nitrogen in the xylem sap of non-nodulated plants grown on nitrogen-free nutrients, but relative levels of this compound decreased and asparagine increased when nitrate was supplied. The presence of 4-methyleneglutamine in xylem exudate did not appear to be associated with either N₂ fixation or nitrate assimilation, and an origin from cotyledon nitrogen was suggested from study of changes in amount of the compound in tissue amino acid pools and in root bleeding xylem sap following germination. Changes in xylem sap composition were studied in nodulated plants receiving a range of levels of ¹⁵N-nitrate, and a ¹⁵N dilution technique was used to determine the proportions of accumulated plant nitrogen derived from N₂ or fed nitrate. The abundance of asparagine in xylem sap and the ratio of asparagine:nitrate fell, while the ratio of nitrate:total amino acid rose as plants derived less of their organic nitrogen from N₂. Assays based on xylem sap composition are suggested as a means of determining the relative extents to which N₂ and nitrate are being used in peanuts.     

Effect of applied nitrate on enzymes of ammonia assimilation in nodules ofCicer arietinum L.

Summary The relationship between N₂-fixation, nitrate reductase and various enzymes of ammonia assimilation was studied in the nodules and leaves ofC. arietinum. In the nodules of the plants growing on atmospheric nitrogen, maximum activities of glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), asparagine synthetase (AS) and aspartate aminotransferase (AAT) were recorded just prior to maximum activity of nitrogenase. In nitrate fed plants, the first major peak of GDH and AS coincided with that of nitrate reductase in the nodules. With the exception of AS, application of nitrate decreased the activities of all these enzymes in nodules but not in leaves. Activities of GS, GOGAT and AAT were affected to much greater extent than that of GDH. On comparing the plants grown without nitrate and those with nitrate, the ratios of the activities of GDH/GS and GDH/GOGAT in nitrate given plants, increased by 4 and 12 fold, respectively. The results presented in this paper suggest that in nodules of nitrate fed plants, assimilation of ammonia via GDH assumes much greater importance.     

Uptake and Distribution of Nitrogen in Young Apple Trees after Application of Nitrate or Ammonium, with Special Reference to Asparagine and Arginine

Apple rootstocks M.7 were given either nitrate or ammonium at the end of September. Until the following June, total and protein nitrogen and the composition of the soluble nitrogen fraction were followed in the roots, stem-bark, and new growth. Nitrogen from both sources was readily absorbed in roughly the same amounts. Absorption occurred in the autumn and especially in the following spring. Incorporation of the absorbed nitrogen took place exclusively in the young roots. Arginine and, to a lower degree, asparagine were by far the most abundant of the soluble amino compounds and reached levels far above those in the unfertilized trees. The asparagine/arginine ratio was rather high during the first few weeks after fertilization but then dropped steadily to a low winter level. During May and June there was a gradual decrease to very low levels, especially in the new growth. The data suggest that the absorbed nitrogen is incorporated into asparagine and that arginine is formed from asparagine if the supply of nitrogen exceeds the immediate needs. Throughout the experimental period, ammonium fertilization led to higher values of the asparagine/arginine ratio than did nitrate nutrition. This is explained in terms of an enhanced production of asparagine and a retarded conversion into arginine. It is argued that asparagine is the main translocation compound for nitrogen. The possibility is discussed that, in addition, arginine moves upwards by a process of exchange along the negatively charged walls of the xylem vessels.     

Distribution of dry weight between root and shoot in white clover dependent on N2 fixation or utilizing abundant nitrate nitrogen

Summary Plants of white clover var. Blanca were grown singly in pots in controlled environments, or in small swards in a glasshouse, to determine how their distribution of dry weight between root and shoot was influenced when they were dependent on N₂ fixation in their root nodules or when they lacked nodules but utilized an abundant supply of nitrate nitrogen. In single plants and in swards, changes in root/shoot ratio with increasing age and plant development were not influenced by the source of nitrogen, but nodulated plants always displayed a higher root/shoot ratio. When nodulated plants were supplied with nitrate nitrogen, root/shoot ratio declined to values intermediate between those of nodulated and of nitrate plants. The results are discussed in relation to the persistence of white clover, and the general level of productivity, in grass-clover swards. The Grassland Research Institute is financed through the Agricultural Research Council.     

Antimicrobial Effect of Red Clover (Trifolium pratense) Phenolic Extract on the Ruminal Hyper Ammonia-Producing Bacterium, Clostridium sticklandii

Ruminal proteolysis and subsequent amino acid degradation represent considerable economic loss in ruminant production. The hyper ammonia-producing bacteria (HAB) are largely responsible for amino acid deamination in the rumen. HAB can be controlled with ionophores, but they are also susceptible to antimicrobial plant secondary metabolites. Red clover (Trifolium pratense) is rich in soluble phenolics, and it is also more resistant to proteolysis than other legumes. The goal of this study was to identify phenolic compounds from Trifolium pratense cultivar Kenland, and determine if any of the compounds possessed antimicrobial activity against the bovine HAB, Clostridium sticklandii SR. HPLC analysis revealed that clover tissues were rich in the isoflavonoids formononetin and biochanin A, particularly in plants left to wilt for 24 h. Biochanin A inhibited C. sticklandii in bioassays that employed thin-layer chromatography (TLC). Both clover extracts and biochanin A inhibited the growth of C. sticklandii in broth culture, but formononetin had no effect. These results indicate that clover phenolic compounds may have a role in preventing amino acid fermentation.     

The Influence of Nitrogen Nutrition on the Accumulation of Free Amino Acids in Root Tissue of Urtica dioica and their Apical Transport in Xylem Sap

Urtica dioica plants were grown on a nitrogen supply of 3, 15and 22 mM with nitrate and ammonium as nitrogen source. In contrastto nitrate reductions amino acid synthesis occurred in roottissue. At 3 mM ammonium obviously the amino acids were rathertransported via xylem upwards to the shoots than stored in theroots. Particularly increased ammonium supply led to stimulatedstorage of free amino acids in the roots, mainly as asparagineand arginine. In xylem asparagine was the dominant nitrogentransporting compound, while arginine was hardly translocated.With the enhancement of nitrogen supply, the second amide, glutamine,became more and more important with respect to the transportof nitrogen. (Received September 3, 1984; Accepted November 2, 1984)     

Haploid and diploid plant regeneration from protoplasts of anther callus in rice

Summary The regeneration of haploid and diploid plants was demonstrated from protoplasts that were isolated from cell suspensions of anther callus in rice. The cell suspension in the AA medium that contained 4 amino acids as the sole nitrogen source was friable, finely dispersed, and readily released a large number of protoplasts. These protoplasts, subsequently cultured in NO₃ medium that contained nitrate as the sole nitrogen source, formed compact calli. The compact calli produced green plants with a frequency of 24%. Out of 15 flowering plants, 4 were haploids, the others were diploids which showed a uniform morphology but varied in seed fertility from 95 to 0%.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid     

Powdery mildew of tobacco (Erysiphe cichoracearum DC.): Susceptibility of proximal and distal parts of leaves from different stalk positions on intact and topped field plants in relation to free amino nitrogen and carbohydrate content*

The area covered by visible mycelium of E. cichoracearum on the upper surface of leaves 4, 8, 12 and 16 of tobacco plants in field plots in Rhodesia was expressed as percentages of the proximal and distal halves at weekly intervals. Free amino nitrogen and carbohydrate in discs from proximal and distal halves of the same leaves were analysed when each leaf was expanding rapidly and was not infected, and several weeks later, when the rate of expansion had slowed down and there was slight infection. On two other occasions, similar leaf discs were inoculated with conidia, to measure the percentage germination and hyphal length from individual conidia after incubation for 2–3 days at constant temperature and humidity; duplicate discs were chemically analysed. Leaves were not susceptible until at least 6 weeks after they had emerged from the bud. Soluble carbohydrate increased and free amino nitrogen decreased during the change from resistance to susceptibility. Proximal parts of leaves were usually infected first; they initially contained less amino nitrogen and soluble carbohydrate than distal parts. All parts of the leaf seemed to be equally susceptible later, when there were no differences in their amino nitrogen or soluble carbohydrate. Upper leaves of intact plants had more natural infection than those from corresponding leaves from topped plants. More conidia germinated on discs from them and produced longer hyphae. The discs from intact plants contained less free amino nitrogen and more soluble carbohydrate than those from topped plants. The accuracy of visual assessments of susceptibility was, generally, confirmed by measurements of percentage germination and length of hyphae from individual conidia on leaf discs. Regressions of hyphal length on leaf composition showed that susceptibility was apparently related inversely to free amino nitrogen and water content and directly to insoluble carbohydrate per unit dry matter.     

Ammonium and nitrate nutrition inPlantago lanceolata L. andPlantago major L. ssp.major

With the aims (1) to test whether the different natural occurrence of twoPlantago species in grasslands is explained by a different preference of the species for nitrate or ammonium; (2) to test whether the different occurrence is explained by differences in the flexibility of the species towards changes in the nitrogen form; (3) to find suitable parameters as a tool to study ammonium and nitrate utilization of these species at the natural sites in grasslands, plants ofPlantago lanceolata andP. major ssp.major were grown with an abundant supply of nitrate, ammonium or nitrate+ammonium as the nitrogen source (0.5 mM). The combination of ammonium and nitrate gave a slightly higher final plant weight than nitrate or ammonium alone. Ammonium lowered the shoot to root ratio inP. major. Uptake of nitrate per g root was faster than that of ammonium, but from the mixed source ammonium and nitrate were taken up at the same rate. In vivo nitrate reductase activity (NRA) was present in both shoot and roots of plants receiving nitrate. When ammonium was applied in addition to nitrate, NRA of the shoot was not affected, but in the root the activity decreased. Thus, a larger proportion of total NRA was present in the shoot than with nitrate alone. In vitro glutamate dehydrogenase activity (GDHA) was enhanced by ammonium, both in the shoot and in the roots.In vitro glutamine synthetase activity (GSA) was highest in roots of plants receiving ammonium. Both GDHA and GSA were higher inP. lanceolata than inP. major. The concentration of ammonium in the roots increased with ammonium, but it did not accumulate in the shoot. The concentration of amino acids in the roots was also enhanced by ammonium. Protein concentration was not affected by the form of nitrogen. Nitrate accumulated in both the shoot and the roots of nitrate grown plants. When nitrate in the solution was replaced by ammonium, the nitrate concentration in the roots decreased rapidly. It also decreased in the shoot, but slowly. It is concluded that the nitrogen metabolism of the twoPlantago species shows a similar response to a change in the form of the nitrogen source, and that differences in natural occurrence of these species are not related to a differential adaptation of nitrogen metabolism towards the nitrogen form. Suitable parameters for establishing the nitrogen source in the field are thein vivo NRA, nitrate concentrations in tissues and xylem exudate, and the fraction of total reduced nitrogen in the roots that is in the soluble form, and to some extent thein vitro GDHA and GSA of the roots. Grassland Species Research Group. Publ. no 118.     

A Threshold Value for Cutting Intensity in Relation to Fusarium Root Rot of Red Clover (Trifolium pratense L.)

Although cutting the foliage is known to increase Fusarium root rot severity in red clover (Trifolium pratense L.), no quantitative relationship has so far been determined. In this study, results from a number of greenhouse experiments, where plants were artificially inoculated with Fusarium avenaceum (Corda ex Fr.) Sacc., show a linear relationship between cutting intensity and Fusarium root rot in red clover, cv. ‘Hermes II’ an increased cutting intensity giving an increased root rot severity. Theoretically, a threshold value can be calculated beyond which no increase in root rot severity, compared to an uncut plant, should be expected.     

Assimilate Partitioning in Red and White Clover Either Dependent on N2 Fixation in Root Nodules or Utilizing Nitrate Nitrogen

During vegetative growth in controlled environments, the patternof distribution of ¹⁴C-labelled assimilates to shoot and root,and to the meristems of the shoot, was measured in red and whiteclover plants either wholly dependent on N₂ fixation in rootnodules or receiving abundant nitrate nitrogen but lacking nodules. In experiments where single leaves on the primary shoot wereexposed to ¹⁴CO₂, nodulated plants of both clovers generallyexported more of their labelled assimilates to root (+nodules),than equivalent plants utilizing nitrate nitrogen, and thiswas offset by reduced export to branches (red clover) or stolons(white clover). The intensity of these effects varied with experiment.The export of labelled assimilate to growing leaves at the terminalmeristem of the donor shoot was not influenced by source ofnitrogen. Internode elongation in the donor shoot utilized nolabelled assimilate. Whole plants of white clover exposed to ¹⁴CO₂ on seven occasionsover 32 days exhibited the same effect on export to root (+nodules),which increased slightly in intensity with increasing plantage. Nodulated plants had larger root: shoot ratios than theirequivalents utilizing nitrate nitrogen. Trifolium repens, Trifolium pratense, red clover, white clover, nitrogen fixation, nitrate utilization, assimilate partitioning     

The Respiratory Costs of Nitrogen Fixation in Soyabean, Cowpea, and White Clover: II. COMPARISONS OF THE COST OF NITROGEN FIXATION AND THE UTILIZATION OF COMBINED NITROGEN

Plants of soyabean, cowpea, and white clover were grown singlyin pots in Saxcil growth cabinets at 23/18 °C, 30/24 °C,and 20/15 °C, respectively, until seed maturation or for85 d (white clover). Two populations were produced within eachspecies: one population effectively nodulated and wholly dependentfor nitrogen on fixation in the root nodules, and a second populationcompletely lacking nodules but receiving abundant nitrate nitrogen.In each species, the two populations were compared in termsof rate of gross photosynthesis, rate of shoot respiration,and rate of root respiration. Source of nitrogen had littleor no effect on rate of photosynthesis or shoot respiration.In contrast, the rate of respiration of the nodulated rootsof plants fixing their own nitrogen was greater, sometimes two-foldgreater, than that of equivalent plants lacking nodules andutilizing nitrate nitrogen. This superiority in terms of rateof root respiration was generally confined to the period ofintense nitrogen fixation. An analysis of the magnitude of thisrespiratory burden in terms of daily photosynthesis indicatesthat, in all three legumes, plants fixing their own nitrogenrespire 11–13% more of their fixed carbon each day thanequivalent plants lacking nodules and utilizing nitrate nitrogen.