The Effect of Reduced Light Intensity and Sub-optimal Potassium Supply on N2 Fixation and N Turnover in Rhizobium Infected Lucerne

The effect of low light intensity and suboptimal potassium supply on the fixation of molecular nitrogen by root nodules and the nitrogen turnover in the host plant was studied in Medicago sativa using ¹⁵N labelled molecular nitrogen. For the application of ¹⁵N₂ labelled gas a special box was used. Both low light intensity and a low potassium supply resulted in a substantial growth depression. In particular the protein content of tops, roots and nodules was lower in the plants of the low light intensity treatment as compared with the control plants. Decreasing potassium supply had a similar but less-pronounced effect on protein content. The low protein content was not a consequence of a lack of soluble amino nitrogen or NH₃, since these fractions were influenced to a lesser degree by the reduced light intensity and by the low potassium supply. This observation is supported by the data obtained with ¹⁵N. N₂ fixation and NH₃ assimilation were affected by both low light intensity and low K application to the same degree as the overall metabolism, thus showing no particular response to the treatments applied.     

Probable site of allantoin formation in nodulating soybean plants

Nodulated soybean plants contain high concentration of allantoin in all parts. Excision of nodules from the roots brought about a marked decrease in allantoin. To examine the function of nodules in allantoin production, nodulated and nodule-detached soybeans were fed with ¹⁵NH₃ for 1 week. High abundance of ¹⁵N was found in the amino acid-N fraction of both plants. In the root and stem of the nodulated plants, ca 80% of the nitrogen in this fraction was derived from the NH₃ added in the medium. Excess ¹⁵N was detected also in allantoin-N fraction, but the ¹⁵N content was very low in contrast to that in amino acid-N fraction. The site involved in the allantoin formation and the possible significance of its synthesis are discussed in relation to symbiotic nitrogen fixation.     

Inorganic carbon fixation and metabolism in maize roots as affected by nitrate and ammonium nutrition

The effects of NO^?₃ and NH⁺₄ nutrition on the rates of dark incorporation of inorganic carbon by roots of hydroponically grown Zea mays L. cv. 712 and on the metabolic products of this incorporation, were determined in plants supplied with NaH¹⁴CO₃ in the nutrient solution. The shoots and roots of the plants supplied with NaH¹⁴CO₃ in the root medium for 30 min were extracted with 80%; (v/v) ethanol and fractionated into soluble and insoluble fractions. The soluble fraction was further separated into the neutral, organic acid, amino acid and non-polar fractions. The amino acid fraction was then analyzed to determine quantities and the ¹⁴C content of its individual components. The rates of dark incorporation of inorganic carbon calculated from H¹⁴CO^?₃ fixation and attributable to the activity of phosphoenolpyuvate carboxylase (EC 4.1.1.31), were 5-fold higher in ammonium-fed plants than in nitrate-fed plants after a 30-min pulse of ¹⁴C. This activity forms a small, but significant component of the carbon budget of the root. The proportion of ¹⁴C located in the shoots was also significantly higher in ammonium-fed plants than in nitrate-fed plants, indicating more rapid translocation of the products of dark fixation to the shoots in plants receiving NH⁺/sp₄ nutrition. Ammonium-fed plants favoured incorporation of ¹⁴C into amino acids, while nitrate-fed plants allocated relatively more ¹⁴C into organic acids. The amino acid composition was also dependent on the type of nitrogen supplied, and asparagine was found to accumulate in ammonium-fed plants. The ¹⁴C labelling of the amino acids was consistent with the diversion of ¹⁴C-oxaloacetate derived from carboxlyation of phosphoenolpyruvate into the formation of both asparatate and glutamate. The results support the conclusion that inorganic carbon fixation in the roots of maize plants provides an important anaplerotic source of carbon for NH⁺₄ assimilation.     

Enriched rhizosphere CO2 concentrations can ameliorate the influence of salinity on hydroponically grown tomato plants

Our previous work indicated that salinity caused a shift in the predominant site of nitrate reduction and assimilation from the shoot to the root in tomato plants. In the present work we tested whether an enhanced supply of dissolved inorganic carbon (DIC, CO₂+ HCO₃) to the root solution could increase anaplerotic provision of carbon compounds for the increased nitrogen assimilation in the root of salinity-stressed Lycopersicon esculentum (L.) Mill. cv. F144. The seedlings were grown in hydroponic culture with 0 or 100mM NaCl and aeration of the root solution with either ambient or CO₂-enriched air (5000 μmol mol^?1). The salinity-treated plants accumulated more dry weight and higher total N when the roots were supplied with CO₂-enriched aeration than when aerated with ambient air. Plants grown with salinity and enriched DIC also had higher rates of NO^?₃ uptake and translocated more NO^?₃ and reduced N in the xylem sap than did equivalent plants grown with ambient DIC. Incorporation of DIC was measured by supplying a 1 -h pulse of H¹⁴CO^?₃ to the roots followed by extraction with 80% ethanol. Enriched DIC increased root incorporation of DIC 10-fold in both salinized and non-salinized plants. In salinity-stressed plants, the products of dissolved inorganic ¹⁴C were preferentially diverted into amino acid synthesis to a greater extent than in non-salinized plants in which label was accumulated in organic acids. It was concluded that enriched DIC can increase the supply of N and anaplerotic carbon for amino acid synthesis in roots of salinized plants. Thus enriched DIC could relieve the limitation of carbon supply for ammonium assimilation and thus ameliorate the influence of salinity on NO^?₃ uptake and assimilation as well as on plant growth.     

The Effect of Darkness on the Leghemoglobin Content and Amino Acid Levels in the Root Nodules of Pea Plants

The dependence of the nitrogen fixing system in the root nodules of pea plants (Pisum sativum) L. cv. Torsdag II) on light induced reactions was studied. The pots of the inoculated pea plants, after the nolules had fixed nitrogen for a fornight, were transferred to a dark room. The control plants were kept under normal lighting conditions. The decay of leghemoglobin was measured after photosynthesis had ceased. In the dark the red nodules turned green in three days, when about half of the haem had been broken down. The plants in normal lighting conditions had maintained the red nodules. The appearence of leghemoglobin and bacteroids was simultaneouos. In normal lighting conditions the number of bacteroids was about 1.6 × 10⁸ per g fresh nodules. The appearance of leghemoglobin and bacteroids was simultaneous. In normal lighting conditons the number of bacteroids was bout 1.6 × 10⁸ per g fresh nodules. At the same time as the nodules turned green in the dark most of the bacteroids disappeared and the number of rod-shaped bacteria increased. After five days int the dark thenumber of bacteria of the green nodules was 2.2 × 10⁸ per g fresh nodules. A large increase of of bacteria in the nodules is one of the results after the termination of effective symbiosis. Quantitative estimations were made with an automatic amino acid analysator of the amino acid composition in the root nodules of pea plants grown in the light and of pea plants grown in the dark. Altogether 27 amino acids and amides and 3 unknown ninhydrin positive compounds were found in the free amino acid fraction. In the red N-fixing nodules asparagine, the amide of aspartic acid, was the most prominent (more than 50 per cent of the total amino acid fraction), indicating the energy charge of the nitrogen fixation. 5 days in the dark affected the proportions of the amino acids as follows. Asparagine, homoserine, γ-aminobutyric acid and ethanolamine were decreased and the most of the others increased. In the hydrolysate of the non-soluble protein fraction 25 amino acids could be detected. The proportions of the amino acids in the root nodules of light-grown and dark-grown pea plants were very similar. Hydroxyproline and α, γ-diaminopimelic acid (DAP) were found in these fraction. Most of the DAP was contained in the peptide fraction. Also hydroxyproline was found to a small extent. It was assumed that the amino acids in this fraction were derived from the peptides of both plant cells and rhizobia.     

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.     

Influence of ammonium and nitrate nitrogen on nitrogenase activity of pea plants as affected by light intensity and sugar addition

Summary Addition of ammonium chloride or potassium nitrate to nodulated pea plants resulted in a decrease in acetylene-reducing activity. Both nodule growth and specific activity of the nodules were diminished. Acetylene-reducing activity of isolated bacteroids, treated with EDTA-toluene and supplied with ATP and dithionite, had not decreased after a 3-day treatment of the plants with NH₄Cl or KNO₃. The effect of combined nitrogen could be counteracted by raising the light intensity or by the addition of sucrose to the growth medium. The latter treatment reduced the nitrogen uptake by the plants. It is concluded that combined nitrogen affects symbiotic nitrogen fixation via the carbohydrate supply to the bacteroids.     

Nitrogen Fixation and Allantoin Formation in Soybean Plants

The activity of nitrogenase and the concentration of ammonia and allantoin (+ allantoic acid) in root nodules were measured throughout the growth period of soybean plants. Nitrogenase activity measured by acetylene reduction increased with plant growth and reached a maximum level at the flowering period. The level of ammonia and allantoin concentration in nodules was parallel with increased nitrogenase activity. At the late reproductive stage (pod-forming period), nitrogenase activity showed a marked decrease, but the ammonia and allantoin in the nodules remained at a constant level. Detached nodules from 56 day-old soybean plants were exposed to ¹⁵N₂ gas, and the distribution of ¹⁵N among nitrogen compounds was investigated. Enrichment of ¹⁵N in allantoin and allantoic acid reached a fairly high level after 90 min of nitrogen fixation; ca. 22% of ¹⁵N in acid-soluble nitrogen compounds was incorporated into allantoin + allantoic acid. In contrast, enrichment of ¹⁵N in amide nitrogen was relatively low. No significant ¹⁵N was detected in the RNA fraction. The data suggested that ureide formation in nitrogen-fixing root nodules did not take place through the breakdown of nucleic acids, but directly associated with the assimilating system of biologically fixed nitrogen.     

Effects of applied nitrogen upon aspects of nitrogen metabolism and transport in developing nodulated winged bean plants

Nodulated winged bean [Psophocarpus tetragonolobus (L.) DC., cv. UPS 122] were grown under constant environmental conditions and supplied with mineral nutrient solution in which nitrogen was absent or was present as nitrate (12 mg N week^-1 plant^-1). Nitrate treatment dramatically promoted plant growth, increased fruit weight 1.6 fold, was necessary for tuberisation and enhanced nodulation. The in vitro accumulation of ¹⁴C into asparagine and aspartate components of excised nodules supplied with exogenous ¹⁴CO₂ and [¹⁴C]-D-glucose was greater for nitrate-treated plants, whilst accumulation into ureides was reduced by nitrate treatment. Levels of amino acids in xylem sap were greater for plants supplied with a complete nutrient solution, than those grown without applied nitrate, particularly for asparagine, glutamine and proline. Xylem ureide levels were greater for plants grown in the absence of supplementary nitrate. Nitrogen accumulated in leaf, stem and petiole, and root nodule tissues for utilisation during fruit development; peak nitrogen levels and time of anthesis were retarded for plants grown without applied nitrate. The shoot ureide content increased during fruiting, coincident with decreases in the total nitrogen content, indicating that ureide pools are not utilised during the early reproductive phase. However ureide reserves, particularly allantoin, were utilised during the later stages of pod fill. Enzyme activity which metabolised asparagine was found throughout the plant and was identified as K⁺-dependent asparaginase (EC 3.5.1.1) and an aminotransferase. Apart from temporal differences in developmental profiles of enzyme activity, the activity of these enzymes and of allantoinase (EC 3.5.2.5) in developing tissues were similar for both treatments. The main differences were greater asparaginase and asparagine:pyruvate aminotransferase activities in root tissues and fruit of nitrate-supplied plants; allantoinase activity in the primary roots of plants grown without nitrate decreased during development, whilst activity in developing tubers (nitrate-supplied plants) increased.     

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     

Primary metabolites of Phaseolus vulgaris nodules

More ethanol soluble material (carbohydrate and amino nitrogen) was found in both host cell and bacteroid components of Phaseolus vulgaris nodules from plants grown at 28 W/m² than from plants grown at 7 W/m². The range of compounds identified was similar at the two irradiances. On feeding ¹⁴CO₂ to the plant tops at either irradiance the labelling patterns of carbohydrates and organic acids in the nodule host cells and bacteroids suggested that any or all of the following substances could be donated by the host to the bacteroids for general metabolism: sucrose, fructose, glucose, an unidentified carbohydrate, malic acid and an organic acid co-chromatographing with 6-phosphogluconate. Distribution and labelling patterns of nodule amino compounds were consistent with the hypothesis that ammonia is the primary product of nitrogen fixation within bacteroids, and that this ammonia is transported to host cells for assimilation, initially into glutamine and glutamate.     

Effects of atrazine on the assimilation of inorganic nitrogen in cereals

The inclusion of sub-lethal amounts ofthe herbicide atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine] in the nutrient solution supplied to maize and barley increased the growth of the root and shoot and the uptake of nitrate. The activities of nitrate and nitrite reductases, glutamine synthetase and glutamate synthase were enhanced and the amino acid and nitrate contents of the xylem sap increased. All these effects of atrazine were found only in plants grown with nitrate as the nitrogen source. The uptake of ¹⁵NO₃^? and its incorporation into protein in the root and shoot of maize and barley seedlings was significantly greater in the atrazine treated plants. However, a stimulation in the incorporation of leucine-[¹⁴C] into TCA-precipitable protein of detached leaves from 7-day-old barley seedlings was obtained only in the absence of a supply of combined nitrogen either in the culture medium or in the in vitro incubation mixture containing the labelled amino acid.     

Effect of shoot removal on remobilization of carbon and nitrogen during regrowth of nitrogen‐fixing alfalfa

The contribution of carbon and nitrogen reserves to regrowth following shoot removal has been studied in the past. However, important gaps remain in understanding the effect of shoot cutting on nodule performance and its relevance during regrowth. In this study, isotopic labelling was conducted at root and canopy levels with both ¹⁵N₂ and ¹³C‐depleted CO₂ on exclusively nitrogen‐fixing alfalfa plants. As expected, our results indicate that the roots were the main sink organs before shoots were removed. Seven days after regrowth the carbon and nitrogen stored in the roots was invested in shoot biomass formation and partitioned to the nodules. The large depletion in nodule carbohydrate availability suggests that root‐derived carbon compounds were delivered towards nodules in order to sustain respiratory activity. In addition to the limited carbohydrate availability, the upregulation of nodule peroxidases showed that oxidative stress was also involved during poor nodule performance. Fourteen days after cutting, and as a consequence of the stimulated photosynthetic and N₂‐fixing machinery, availability of C_new and N_new strongly diminished in the plants due to their replacement by C and N assimilated during the post‐labelling period. In summary, our study indicated that during the first week of regrowth, root‐derived C and N remobilization did not overcome C‐ and N‐limitation in nodules and leaves. However, 14 days after cutting, leaf and nodule performance were re‐established.     

Transport and Distribution of 14CO2 Assimilate in Lolium perenne in Response to Varying Nitrogen Supply to Halves of a Divided Root System

The movement of ¹⁴C assimilate from shoots to roots and its subsequent metabolism in the root of Lolium perenne L. was studied using variable N nutrition supplied to halves of a divided root system. Half of the N-deficient root system was supplied with either high NO₃-N or high NH₄-N for 16 hours or 6 days before ¹⁴CO₂ labeling of the shoots. The distribution of ¹⁴C in sugars, ethanol-soluble nitrogen and organic acids in roots appeared to be related to the N content of the tissue. Supply of high NO₃-N for 6 days resulted in significant internal translocation of N into the low N supplied root half. Both root halves also had similar patterns of ¹⁴C distribution among soluble and insoluble metabolites. However, NH₄-N supply for 6 days did not result in a significant increase of N in the low N supplied roots, thus only the high NH₄-N supplied roots displayed stimulated sugar metabolism, similar to that in both root halves in the high NO₃-N supply treatment. Percent transport of ₁₄C assimilates from shoot to root was influenced by form and level of N supplied to root halves. Root halves supplied with either high N source for 6 days accumulated greater amounts of ¹⁴C assimilate than the corresponding low N root half. However NH₄-N supply appeared to make roots stronger sinks since NH₄ supply resulted in significantly greater ¹⁴C accumulation in both the high NH₄ supplied and the low N root halves than did NO₃-N supply in corresponding root halves. The data suggest that factors other than root metabolism, such as N mediated metabolism in the shoot, may also influence the percent transport of assimilates to the root. Internal distribution of the incoming assimilate within the root system could be regulated by the metabolic activity or assimilate demand of the roots.     

Carbohydrate supply and n(2) fixation in soybean : the effect of varied daylength and stem girdling

When arrival of shoot supplied carbohydrate to the nodulated root system of soybean was interrupted by stem girdling, stem chilling, or leaf removal, nodule carbohydrate pools were utilized, and a marked decline in the rates of CO₂ and H₂ evolution was observed within approximately 30 minutes of treatment. Nodule excision studies demonstrated that the decline in nodulated root respiration was associated with nodule rather than root metabolism, since within 3.5 hours of treatment, nodules respired at less than 10% of the initial rates. Apparently, a continuous supply of carbohydrate from the shoot is required to support nodule, but not root, function. Depletion of nodular carbohydrate pools was sufficient to account for the (diminishing) nodule respiration of girdled plants. Of starch and soluble sugar pools within the whole plant, only leaf starch exhibited a diurnal variation which was sufficient to account for the respiratory carbon loss of nodules over an 8 hour night. Under 16 hour nights, or in continuous dark, first the leaf starch pools were depleted, and then nodule starch reserves declined concomitant with a decrease in the rates of CO₂ and H₂ evolution from the nodules. Nodule soluble sugar levels were maintained in dark treated plants but declined in girdled plants. The depletion of starch in root nodules is an indicator of carbohydrate limitation of nodule function.     

Mode of action of chlorsulfuron in a sensitive wheat (Triticum aestivum) cultivar: primary and secondary effects on nitrogen assimilation

Chlorsulfuron (15 g a.i. ha^-1) inhibited growth of wheat (Triticurn aestivum L. cv. Rongotea) especially on high nitrate (NO₃) supply. Decreased growth at high NO^-₃ was associated with higher concentrations of reduced nitrogen (N) and NO^-₃ in the shoots. Seven days after spraying (DAS), shoot dry weight (dry wt) of sprayed plants was similar with NO^-₃ or branched chain amino acids as main N supply but 28 DAS, shoot dry wt was greater with the amino acid treatment. One DAS, chlorsulfuron caused substantial decreases in extension of the youngest leaf and acetolactate synthase activity and valine content of shoots of plants supplied with NO^-₃ or branched chain amino acids. Total amino acid content of shoots was greater in sprayed plants than in unsprayed plants 1 DAS. Acetolactate synthase activity of sprayed plants supplied low NO^-₃ returned to normal 14–21 DAS. For sprayed plants transferred from low to high NO^-₃ supply 7, 14 or 21 DAS, shoot dry wt 50 DAS increased with increased time of transfer to high NO^-₃ while shoot NO^-₃ content decreased. Shoot NO₃ content of sprayed plants transferred to high NO^-₃ supply 7 or 14 DAS was similar to that in unsprayed plants at applied NO^-₃ concentrations which inhibited growth. It is concluded that inhibition of acetolactate synthase is likely to be the primary mode of action of chlorsulfuron in this wheat cultivar; data are consistent with the proposal that subsequent NO^-₃ accumulation can also inhibit growth.     

Transport and Partitioning of CO(2) Fixed by Root Nodules of Ureide and Amide Producing Legumes

Nodulated and denodulated roots of adzuki bean (Vigna angularis), soybean (Glycine max), and alfalfa (Medicago sativa) were exposed to ¹⁴CO₂ to investigate the contribution of nodule CO₂ fixation to assimilation and transport of fixed nitrogen. The distribution of radioactivity in xylem sap and partitioning of carbon fixed by nodules to the whole plant were measured. Radioactivity in the xylem sap of nodulated soybean and adzuki bean was located primarily (70 to 87%) in the acid fraction while the basic (amino acid) fraction contained 10 to 22%. In contrast, radioactivity in the xylem sap of nodulated alfalfa was primarily in amino acids with about 20% in organic acids. Total ureide concentration was 8.1, 4.7, and 0.0 micromoles per milliliter xylem sap for soybean, adzuki bean, and alfalfa, respectively. While the major nitrogen transport products in soybeans and adzuki beans are ureides, this class of metabolites contained less than 20% of the total radioactivity. When nodules of plants were removed, radioactivity in xylem sap decreased by 90% or more. Pulse-chase experiments indicated that CO₂ fixed by nodules was rapidly transported to shoots and incorporated into acid stable constituents. The data are consistent with a role for nodule CO₂ fixation providing carbon for the assimilation and transport of fixed nitrogen in amide-based legumes. In contrast, CO₂ fixation by nodules of ureide transporting legumes appears to contribute little to assimilation and transport of fixed nitrogen.     

Utilization of exogenously supplied 14C-saccharose into primary metabolites and alkaloid production in Catharanthus roseus

Partitioning of exogenously supplied U-¹⁴C-saccharose into primary metabolic pool as sugars, amino acids, and organic acids was analyzed and simultaneous utilization for production of alkaloid by leaf, stem, and root in twigs and rooted plants of Catharanthus roseus grown in hydroponic culture medium was determined. Twigs revealed comparable distribution of total ¹⁴C label in leaf and stem. Stems contained significantly higher ¹⁴C label in sugar fraction and in alkaloids [47 kBq kg⁻¹(DM)] than leaf. In rooted plants, label in ¹⁴C in metabolic fractions in root such as ethanol-soluble, ethanol-insoluble, and chloroform-soluble fractions and in components such as sugars, amino acids, and organic acids were significantly higher than in stems and leaves. This was related with significantly higher content of ¹⁴C in alkaloids in stems and leaves. ¹⁴C contents in sugars, amino acids, and organic acids increased from leaf to stem and roots. Roots are the major accumulators of metabolites accompanied by higher biosynthetic utilization for alkaloid accumulation.     

Asparagine as a major factor in the N‐feedback regulation of N2 fixation in Medicago truncatula

The objective of this study was to assess whether a whole plant N‐feedback regulation impact on nitrogen fixation in Medicago truncatula would manifest itself in shifts of the composition of the amino acid flow from shoots to nodules. Detected shifts in the phloem amino acid composition were supposed to be mimicked through artificial phloem feeding and concomitant measurement of nodule activity. The amino acid composition of the phloem exudates was analyzed from plants grown under the influence of treatments (limiting P supply or application of combined nitrogen) known to reduce nodule nitrogen fixation activity. Plants in nutrient solution were supplied with sufficient (9 µM) control, limiting (1 µM) phosphorus or 3 mM NH₄NO₃ (downregulated nodule activity). Low phosphorus and the application of NH₄NO₃ reduced per plant and specific nitrogenase activity (H₂ evolution). At day 64 of growth, phloem exudates were collected from cuts of the shoot base. The amount of amino acids was strongly increased in both phloem exudates and nodules of the treatments with downregulated nodule activity. The increase in the downregulated treatments was almost exclusively the result of a higher proportion of asparagine in both phloem exudates and nodules. Leaf labeling with ¹⁵N showed that nitrogen from the leaves is retranslocated to nodules. An artificial phloem feeding with asparagine resulted in an increased concentration of asparagine in nodules and a decreased nodule activity. A possible role of asparagine in an N‐feedback regulation of nitrogen fixation in M. truncatula is discussed.     

Ammonium effects on function and structure of nitrogen-fixing root nodules of Alnus incana (L.) Moench

Cloned plants of Alnus incana (L.) Moench were inoculated and grown without combined nitrogen for seven weeks. The effects of ammonium on the function and structure of the root nodules were studied by adding 20 mM NH₄Cl (20 mM KCl=control) for four days. Nitrogenase activity decreased to ca. 50% after one day and to less than 10% after two days in ammonium treated plants, but was unaffected in control plants. The results were similar at photon flux densities of 200 and 50 mol m^-2 s^-1. At the higher light level the effect was concentration dependent between 2 and 20 mM NH₄Cl. The recovery was slow, and more than 11 d were needed for plants treated with 20 mM ammonium to reach initial activity. The distribution of ¹⁴C to the root nodules after assimilation of ¹⁴CO₂ by the plants was not changed by the ammonium treatment. Microscopical studies of root nodules showed high frequencies of endophyte vesicles being visually damaged in nodules from ammonium-treated plants, but not in nodules from control plants. When nitrogenase activity was restored, visually damaged vesicles were again few, whereas young developing vesicles were numerous. The slow recovery, the ¹⁴C-translocation pattern, and the structural changes of the endophyte indicate a more complex mechanism of ammonium influence than simply a short-term reduction in supply of carbon compounds to the nodules.