A kinetic study of the assimilation of 15N-labelled nitrate in rice seedlings

A ¹⁵N kinetic-analysis of the assimilation of nitrate nitrogenin the roots of rice seedlings indicated that (1) nitrate wasrapidly reduced to ammonia in the roots, where it was incorporatedinto glutamine and glutamic acids; (2) the pattern of nitrateassimilation into amino acids was very similar to that of ammoniumassimilation; and (3) the pattern of nitrogen incorporationinto protein was also similar to that of the incorporation withNH₄-feeding. In the shoots, alanine, serine, glutamic acid, -amino butyricacid and aspartic acid were relatively strongly labelled with¹⁵N as compared with the other amino acids. A different mechanismof nitrogen assimilation seems to operate in between the photosyntheticand non-photosynthetic organs of the plants. (Received August 19, 1974; )     

Evidence of ammonium assimilation via the glutamine synthetase-glutamate synthase system in rice seedling roots

When rice seedling roots were fed ¹⁵N-ammonium for 1 hr, theamide nitrogen of glutamine showed the highest ¹⁵N abundance.Moreover, glutamine amino, glutamic acid, aspartic acid andalanine showed higher ¹⁵N abundance than ammonium did. In roots whose GS activity was inhibited with MS, both the amountof ammonium and its ¹⁵N abundance were increased. In contrast,both the amount of all examined amino acids containing glutamicacid and their ¹⁵N abundance decreased in roots whose GS activitywas inhibited. From these results, it could be concluded thatthe first step of ammonium assimilation in rice seedling rootswas mainly glutamine synthesis by GS and the second was glutamicacid formation by the GOGAT system. The results of an experiment using ¹⁵N glutamine also supportedthis conclusion. (Received February 23, 1977; )     

The Significance of Transpirationally Derived Nitrogen in Protein Synthesis in Fruiting Plants of Pea (Pisum sativum L.)

Feeding of ¹⁵N-nitrate, ¹⁵N(amide)-L-glutamine, or ¹⁵N-L-glutamicacid to detached shoots of pea through the transpiration streamresults in the soluble and insoluble nitrogen of stem, leaves,and fruits becoming extensively enriched with isotopic nitrogen.The time course of labelling suggests that non-reproductiveparts are the principal centres of uptake and assimilation andthat from them translocation takes place to the developing seeds. Distribution patterns for ¹⁵N in free and protein-bound aminoacids of leaf and seed indicate that each labelled source donatesnitrogen to a wide range of amino compounds, with no evidenceof consistent differences in the manner in which each is assimilated.Alanine, glutamic acid, homoserine, and -aminobutyric acid,are the main recipients of ¹⁵N in the soluble fraction of theleaves, whilst in the insoluble fraction nitrogen of the aminoacids serine, glycine, alanine, threonine, glutamic acid + glutamine,and aspartic acid + asparagine achieves high specific labelling.Amino acids of the seeds are labelled more uniformly with ¹⁵N. A complementary ¹⁴C-labelling experiment on the translocationof photosynthetically fixed carbon from leaf to seed is describedand the labelling patterns obtained for amino acids in leaf,seed, and phloem exudate are discussed in relation to thosefor ¹⁵N.     

Assimilation and transport of nitrogen in rice II. 15N-labelled nitrate nitrogen

An investigation was made to study the assimilation and transportof ¹⁵N-labelled nitrate nitrogen in rice plant (Oryza sativaL.). Nitrogen from labelled nitrate at the end of plant feedingwas found mainly in nitrate form, and was more prevalent inroots, stem and leaf sheaths. The nitrite fraction had the nextlargest ¹⁵N enrichment. The ¹⁵NO₃ assimilation in the newlyemerged panicle was mainly in amide and amino acid. The ¹⁵N-incorporation at day 0 was greatest in amino acid andnitrate of roots and decreased towards the stem and leaves.Incorporation in these fractions considerably decreased fromday 0 to day 10. Probably most of the nitrogen from the nitratesource was transported from the roots to the shoot in nitrateand amino acid forms. A decrease of ¹⁵N-incorporation in the soluble N fraction andincrease in the insoluble N fraction from day 0 to day 10 inplant parts, particularly the blades, suggested that proteinsynthesis occurred mostly in young parts of the shoot duringthis period. The marked variation in ¹⁵N distribution in differentparts of the plant during the 10 days indicated that the nitrogenin roots and tillers was probably remobilized and transportedto other parts, particularly the upper leaf blades. Ammonium and nitrate nitrogen transport in rice plant are compared. (Received May 11, 1974; )     

Assimilation of Nitrogen in Mutants Lacking Enzymes of the Glutamate Synthase Cycle

¹⁵N labelling was used to investigate the pathway of nitrogenassimilation in photorespiratory mutants of barley (Hordeumvulgare cv. Maris Mink), in which the leaves have low levelsof glutamine synthetase (GS) or glutamate synthase, key enzymesof ammonia assimilation. These plants grew normally when maintainedin high CO₂, but the deletions were lethal when photorespirationwas initiated by transfer to air. Enzyme levels in roots weremuch less affected, compared to leaves, and assimilation oflabelled nitrate into amino acids of the root showed very littledifference between wild type and mutants. Organic nitrogen wasexported from roots in the xylem sap mainly as glutamine, levelsof which were somewhat reduced in the GS-deficient mutant andenhanced in the glutamate synthase deficient mutant. In theleaf, the major effect was seen in the glutamatesynthase mutant,which had an extremely limited capacity to utilize the importedglutamine and amino acid synthesis was greatlyrestricted. Thiswas confirmed by the supply of [¹⁵N]-glutamine directly to leaves.Leaves of the GS-deficient mutant assimilatedammonia at about75% the rate found for the wild type, and this was almost completelyeliminated by addition of the inhibitormethionine sulphoximine.Root enzymes, together with residual levels of the deleted enzymesin the leaves, have sufficient capacityfor ammonia assimilation,through the glutamate synthase cycle, to provide adequate inputof nitrogen for normal growth of themutants, if photorespiratoryammonia production is suppressed. Key words: Hordeum vulgare, ¹⁵N, glutamine synthetase, glutamate synthase, ammonia assimilation     

Assimilation and transport of nitrogen in rice I. 15N-labelled ammonium nitrogen

The assimilation and transport of ¹⁵N-labelled ammonium nitrogenin rice plants (Oryza sativa L.) was studied. Plants assimilatedlarge amounts of nitrogen from labelled ammonium into theiramides and amino acids, particularly in the roots and stem,at the end of a 4-day ¹⁵N feeding and 10 days later in the upperleaves, especially in the blades. Although the incorporationof ¹⁵N into all the nitrogen fractions of the newly emergedpanicle was evident, it was particularly pronounced in the amidesand amino acids of the soluble fractions. The upper leaves hada greater ¹⁵N incorporation in their organic N-fractions thandid the lower ones. Amides and amino acids are considered tobe the main forms of nitrogen transported to the shoot fromthe ammonium assimilated in the roots. The transport of theorganic forms of nitrogen was possibly greater to the upperleaves than to the lower ones. The nitrite fraction had more ¹⁵N than did the nitrate fractionin all parts of the plant, particularly in the upper leaf blades.It appeared that some of the ammonia might have been oxidizedto nitrite, then to nitrate in some parts of the plant; probablyin the upper leaves. The synthesis of protein and nucleic acid occurred rapidly inthe upper leaves, especially in the blades, also in the rootsas evidenced by the considerable incorporation of ¹⁵N in theinsoluble fractions of these parts. The variation in ¹⁵N-distribution,during the 10 days, in the different plant parts suggests thatthe nitrogen incorporated during protein synthesis in the rootsand tillers was remobilized and transported to the upper partsof the shoot. A concept for the transport of organic nitrogenouscompounds from the roots to shoot through the phloem and xylemof the rice plant stem is discussed. (Received May 11, 1974; )     

Impact of UV radiation on the production patterns and composition of dissolved free and combined amino acids in marine phytoplankton

Using the ¹³C tracer technique in conjunction with gas chromatographic-massspectro-metric (GC-MS) techniques, we examined the patternsof synthesis and the composition of dissolved free and combinedamino acids within phytoplankton photosynthesizing in the presenceand absence of natural solar ultraviolet radiation (UVR). Atlevels that still permitted the uptake of carbon assimilationinto the cells, UVR caused a marked decline in the overall rateof carbon incorporated into amino acids and a reduction in thepool size of total cellular amino acids (TCAA). In contrast,absolute concentrations of amino acids within the intracellulardissolved free amino acid (INDFAA) pool (measured using an aminoacid analyzer) were higher in the presence of UVR. An examinationof the production patterns and composition of amino acids constitutingthe INDFAA and TCAA pools revealed a marked diminution in thesynthesis and accumulation of alanine and valine in the presenceof UVR. On the other hand, the rates of synthesis and concentrationsof glutamic acid (glutamic acid + glutamine) in the INDFAA andTCAA pools of phytoplankton were higher in samples exposed toUVR. These changes are discussed with reference to the knowneffects of UVR on nitrogen and carbon assimilation within phytoplankton.     

The Route of Nitrate-Nitrogen Assimilation in the Root of Datura stramonium L.

Datura roots were pressure-infiltrated with 400 µg ml^–115N-nitrate feeding solutions with and without the additionof 7 mM L-methionine-DL-sulphoximine (MSO), a glutamine synthetaseinhibitor. Over a 30 min time course the main diversion of newlyreduced ¹⁵N in MSO untreated roots was to glutamine. In MSO-treatedroots ammonia assimilation into amino compounds was completelysuppressed, with resultant accumulation of a large ¹⁵N ammoniapool. This treatment also caused marked concentrational changesin the free amino compound pools, suggesting that conditionsof nitrogen stress had been induced. Glutamate dehydrogenaseactivity was unaffected by the MSO treatment. The results are consistent with the concept that the glutaminesynthetase/glutamate synthase pathway is the major route ofnewly reduced nitrogen assimilation in Datura roots.     

Amino acid metabolism in plant leaf IV. The effect of light on ammonium assimilation and glutamine metabolism in the cells isolated from spinach leaves

Comparison of incorporation of ¹⁵N-labeled ammonium into aminoacids in cells isolated from spinach leaves showed that ammoniumwas most actively incorporated into the amido-group of glutamine.The ¹⁵N contents of other amino acids were less than one-tenththat of the amido-group of glutamine. L-Methionine-DL-sulfoximine(MS) suppressed the incorporation of ammonium not only intothe amido-group of glutamine, but also into glutamic acid. Turningoff the light after 1 min illumination increased die 15N contentof glutamine while it decreased that of the glutamic acid, asparticacid and alanine. Illumination of the cells after die applicationof ammonium had a more significant effect on ammonium assimilationthan illumination before the application of ammonium. When ¹⁴C-U-¹⁵N(amido labeled)-glutamine was added to the cell suspension,the transfer of amido-group of glutamine was completely inhibitedin the dark, but no difference in the flow of ¹⁴C was observed. These results suggest that glutamine synthetase (GS) and glutamatesynthase (GOGAT) pathways operate in ammonium assimilation inthe cells isolated from spinach leaves, and that the formeris light-independent but die latter is light-dependent. (Received December 23, 1977; )     

Influence of Several Growth Regulators and Amino Acids on in vitro Organogenesis of Torenia fournieri Lind.

The effects of several growth regulators and amino acids onin vitro organogenesis of Torenia fournieri Lind. were determinedusing internodal segments. Treatment with 2,4-D¹ resulted innodular callus formation, while NAA and IAA induced roots constantlybut much less frequently shoot buds. Individually BA, zeatin,and 4-PU induced bud formation, but these shoot buds did notdevelop further. Formation of buds by cytokinin was influencedby a simultaneous application of NAA or 2,4-D, but not of IAA,its degree being reduced when BA was simultaneously appliedwith NAA or 2,4-D. When zeatin or kinetin was added with NAA,numerous roots were induced. The effects of various L-amino acids on in vitro organogenesiswere also investigated using the defined medium in which KNO₃was a principal source of nitrogen. The formation of buds wasconsiderably stimulated by alanine and asparagine, and slightlyby glutamic acid in the medium containing both NAA and BA, inwhich bud formation was easily induced. On the other hand, allamino acids except for glutamic acid and aspartic acid inhibitedroom formation in this medium. Root formation was greatly stimulated by proline, alanine, glutamine,glutamic acid, and aspartic acid, and slightly by arginine andtryptophan in the medium containing NAA but no BA. Glutamicacid and aspartic acid also enhanced bud formation in this medium.     

Origin of Foliar Nitrogen and Changes in Free Amino-Acid Composition and Content of Leaves, Stubble, and Roots of Perennial Ryegrass During Re-growth after Defoliation

Nitrogen re-mobilization and changes in free amino acids werestudied as a function of time in leaves, stubble, and rootsduring ryegrass (Lolium perenne L.) re-growth. Experiments with¹⁵N labelling clearly showed that during the first days nearlyall the nitrogen in new leaves came from organic nitrogen re-mobilizedfrom roots and stubble. On the days of defoliation, stubblehad the highest content of free amino acids with 23 mg per gdry weight against 15 mg and 14 mg in leaves and roots, respectively.The major amino acids in leaves were asparagine (23% of totalcontent in free amino acids), aminobutyrate, serine, glutamine,and glutamate (between 7% and 15%) whereas in roots and stubblethe contribution of amides was high, especially asparagine (about50%). Re-growth after cutting was associated with a rapid increaseof the free amino acid content in leaves, with a progressivedecrease in roots while stubble content remained virtually unchanged.In leaves, asparagine increased from the first day of re-growth,while the aspartate level remained unchanged and glutamine increasedstrongly on the first day but decreased steadily during thenext few days of re-growth. Asparagine in stubble and rootschanged in opposite directions: in stubble it tended to increasewhereas in roots it clearly decreased. In contrast, stubbleand roots showed a similar decrease in glutamine. In these twoplant parts, as in leaves, aspartate remained at a low level.Results concerning free amino acids are discussed with referenceto nitrogen re-mobilization from source organs (stubble androots) to the sink organ (regrowing leaves). Key words: Lolium perenne L, re-growth, nitrogen, free amino acids, glutamine, asparagine     

Dynamics of Carbon Photosynthetically Assimilated in Nodulated Soya Bean Plants under Steady-state Conditions 2. The Incorporation of 13C into Carbohydrates, Organic Acids, Amino Acids and some Storage Compounds

Nodulated soya bean (Glycine max L.) plants at the early floweringstage were allowed to assimilate ¹³CO₂ under steady-state conditions,with a constant ¹³C abundance, for 8 h in the light. The plantswere either harvested immediately or 2 d after the end of the¹³CO₂ feeding, divided into young leaves (including flower buds),mature leaves, stems+petioles, roots and nodules; the ¹³C abundancein soluble carbohydrates, organic acids, amino acids, starchand poly-ß-hydroxybutyric acid was determined witha gas chromatography-mass spectrometry. The rapid turnover of ¹³C in the sucrose pools observed in allorgans of the plants showed that sucrose was the principal materialin the translocation stream of primary products of photosynthesis.At the end of the ¹³CO₂ exposure, sucrose in the mature leavesas the major source organs and in the stems+petioles was labelledwith currently assimilated carbon to about 75 per cent, whereasa much higher labelling of sucrose was found in the roots andin the nodules. This suggests the existence of two or more compartmentedpools of sucrose in mature leaves and also in stems+petioles. The relative labelling patterns of individual organic acidsand amino acids were similar in various plant organs. However,the rapid turnover of succinate and glycine was characteristicof nodules. Treatment with a high concentration of nitrate inthe nutrient media increased the turnover rate of amino acidcarbon in shoot organs and roots, while it markedly decreasedthe labelling of amino acids in nodules. The cyclitols, exceptfor D-pinitol, were significantly labelled with assimilated¹³C in mature leaves, but in nodules, the labelling was verymuch less. In the nodules, which were actively fixing atmospheric nitrogen,a large proportion (80–90 per cent) of currently assimilatedcarbon was found as sucrose and starch at the end of the ¹³CO₂feeding. This was also true of the roots. On the other hand,in young growing leaves, the distribution of currently assimilatedcarbon into sucrose, starch and other soluble compounds wasmuch less. This suggests that a large amount of carbon assimilatedby and translocated to young leaves was used to make up structuralmaterials, mainly protein and cell wall polymers synthesis,during the light period. Glycine max L., soya bean, ¹³CO₂ assimilation, carbon metabolism in nodules     

Excretion products of maize roots from seedling to seed development stage

Summary Excretion products of maize roots (Zea mays cv. Koshu) were estimated. All excreted products were the highest by fresh weight basis at the young seedling stage. In amino acids excreted, glutamic acid accounting for 60% of the total was the highest and followed by alanine. These two amino acids showed the diverse fluctuation according to the growing age, that is, glutamic acid increased while alanine decreased. Stachyose was a main soluble sugar excepting the stage prior to the heading. At this stages, glucose and fructose together with stachyose were observed. Lactic acid was the most dominant organic acid through whole growing stages. These excreted materials could be positive factors for the growth ofSpirillum lipoferum which can fix nitrogen non-symbiotically at the rhizosphere of maize.     

Ammonia Assimilation in Zea mays L. Infected with a Vesicular-Arbuscular Mycorrhizal Fungus Glomus fasciculatum

To investigate nitrogen assimilation and translocation in Zea mays L. colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum (Thax. sensu Gerd.), we measured key enzyme activities, 15N incorporation into free amino acids, and 15N translocation from roots to shoots. Glutamine synthetase and nitrate reductase activities were increased in both roots and shoots compared with control plants, and glutamate dehydrogenase activity increased in roots only. In the presence of [15N]ammonium, glutamine amide was the most heavily labeled product. More label was incorporated into amino acids in VAM plants. The kinetics of 15N labeling and effects of methionine sulfoximine on distribution of 15N-labeled products were entirely consistent with the operation of the glutamate synthase cycle. No evidence was found for ammonium assimilation via glutamate dehydrogenase. 15N translocation from roots to shoots through the xylem was higher in VAM plants compared with control plants. These results establish that, in maize, VAM fungi increase ammonium assimilation, glutamine production, and xylem nitrogen translocation. Unlike some ectomycorrhizal fungi, VAM fungi do not appear to alter the pathway of ammonium assimilation in roots of their hosts.     

Salinity Stress and the Content of Proline in Roots of Pisum sativum and Tamarix tetragyna

Amino acid composition of the free amino acid pool and the TCA-insolubleprotein fraction were investigated in root tips of pea and Tamarixtetragyna plants grown at various levels of NaCl salinity. Salinitystress induced an increase of proline content, mainly in thefree amino acid pool in both plants, and of proline or hydroxyprolinecontent in the protein. Externally-supplied proline was absorbedand incorporated into protein, by pea roots, more effectivelythan by Tamarix roots. Salinity stress, apparently, stimulatedthe metabolism of externally-supplied labelled proline. Pearoots have a very large pool of free glutamic acid; however,70 per cent of the ¹⁴C from externally-supplied ¹⁴C-U-glutamicacid was released as CO₂. Very small amounts of it were incorporatedinto protein. No measurable amount of radioactivity could bedetected in any one of the individual amino acids, either ofprotein hydrolysate or the free amino acid pool. Proline very effectively counteracted the inhibitory effectof NaCl on pea seed germination and root growth. A similar effectbut to a lesser degree was achieved with phenylalanine and asparticacid. The feasibility of proline being a cytoplasmic osmoticumis discussed.     

Nitrogen assimilation in citrus trees

Assimilation of ¹⁵N-ammonium and ¹⁵N-nitrate was examined in 3-year-old satsuma mandarin (Citrus unshiu Marcovitch) trees. Experiments were designed to establish the time course of incorporation of nitrogen just taken up into amino compounds. In fine roots, absorbed ¹⁵N-ammonium was actively incorporated into glutamine and then into glutamic acid and asparagine. When feeding ¹⁵N-nitrate, glutamic acid and asparagine were actively synthesized, but glutamine synthesis was comparatively low as compared with that in ammonium feeding. In current leaves and fruits, a clear difference in the labelling patterns of amino acids was found between the ammonium and nitrate feedings. The amino acid most markedly labelled was asparagine in the ammonium feeding and glutamine in the nitrate feeding. Considering the most heavily labelled component in leaves and fruits, the main form of the nitrogen components transported upward in the xylem was discussed.     

Investigation on the Assimilation of Nitrogen by Maize Roots and the Transport of Some Major Nitrogen Compounds by Xylem Sap

The amino acid and protein metabolism of roots of maize has been studied. The important role of the free amino acids and proteins of the roots as active agents in nitrogen assimilation is pointed out. Nitrogen supplied as nitrate is preferably incorporated into α-ketoglutaric acid, and then by trans-aminases transferred to other ketoacids. In the case of ammonia supply the function of a nitrogen-accumulating assimilation system leading to the formation of Arg, Glu-NH₂ and Asp-NH₂ is shown.     

Amino Acid metabolism of pea leaves: diurnal changes and amino Acid synthesis from N-nitrate

In the young leaves of pea (Pisum sativum L.) plants, there was a diurnal variation in the levels of amino acids. In the light, total amino nitrogen increased for the first few hours, then stabilized; in the dark, there was a transient decrease followed by a gradual recovery. Asparagine, homoserine, alanine, and glutamine accounted for much of these changes. The incorporation of ¹⁵N into various components of the young leaves was followed after supply of ¹⁵N-nitrate. ¹⁵N appeared most rapidly in ammonia, due to reduction in the leaf, and this process took place predominantly in the light. A large proportion of the primary assimilation took place through the amide group of glutamine, which became labeled and turned over rapidly; labeling of glutamic acid and alanine was also rapid. Asparagine (amide group) soon became labeled and showed considerable turnover. Slower incorporation and turnover were found for aspartic acid, γ-aminobutyric acid, and homoserine. Synthesis and turnover of all of the amino acids continued at a low rate in the dark. γ-Aminobutyric acid was the only compound found to label more rapidly in the dark than in the light.     

Aspects of the Drought Tolerance in Creosotebush (Larrea divaricata)

In order to understand better the physiological adaption of creosotebush (Larrea divaricata Cav.) to drought conditions, its carbohydrate and nitrogen metabolism after a 7-day desiccation period under controlled conditions were studied. Although fructose was not significantly altered in the leaves of desiccated plants, as compared to those maintained under normal moisture conditions, both glucose and sucrose were significantly reduced. Total amino acids more than doubled under moisture stress, the increase being predominantly due to proline, phenylalanine, and glutamic acid. Significant increases also occurred in alanine, arginine, histidine, isoleucine, and valine. Increases or decreases in other amino acids were not significant. These stress-induced changes in certain amino acids are considered in relationship to protein hydrolysis, to accumulation of nitrogen degradation products translocated from the roots, and to the possible function of specific amino acids (e.g., proline) in NH₃⁺ storage.     

Phosphate Regulation of Nitrate Assimilation in Soybean

It is known that phosphorus deficiency results in alterationsin the assimilation of nitrogen. An experiment was conductedto investigate mechanisms involved in altered ¹⁵NO^–₃ uptake,endogenous ¹⁵N translocation, and amino acid accumulation insoybean (Glycine max L. Merrill, cv. Ransom) plants deprivedof an external phosphorus supply for 20 d in solution culture.Phosphorus deprivation led to decreased rates of ¹⁵NO^–₃uptake and increased accumulation of absorbed ¹⁵N in the root.Both effects became more pronounced with time. Asparagine, theprimary transport amino acid in soybean, accumulated in largeexcess in roots and stems. In roots of phosphorus-deprived plants,concentrations of ATP and inorganic phosphate declined rapidly,but dry weight accumulation was similar to or above that ofthe control even after 20 d of treatment. Arginine accumulationin leaves was greatly enhanced, even though ¹⁵N partitioninginto the insoluble reduced-N fraction of leaves was unaffected.The results suggest that decreases in NO^–₃ uptake in lowphosphorus plants could be caused by feedback control factorsand by limited ATP availability. The decline in endogenous Ntransport from the root to the shoot may be associated withchanges in membrane properties, which also result in paralleleffects on hydraulic conductance and the upward flow of waterthrough the plant. Key words: Phosphorus stress, nitrate uptake, nitrate translocation, arginine