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     

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.     

Effect of localized nitrogen availability to soybean half-root systems on photosynthate partitioning to roots and nodules

Soybean (Glycine max [L.] Merr. cv Davis) was grown in a split-root growth system designed to maintain control of the root atmosphere. Two experiments were conducted to examine how 80% Ar:20% O₂ (Ar:O₂) and air (Air) atmospheres affected N assimilation (NH₄NO₃ and N₂ fixation) and the partitioning of photosynthate to roots and nodules. Application of NH₄NO₃ to nonnodulated half-root systems enhanced root growth and root respiration at the site of application. A second experiment applied Ar:O₂ or air to the two sides of nodulated soybean half-root systems for 11 days in the following combinations: (a) Air to both sides (Air/Air); (b) Air to one side, Ar:O₂ to the other (Air/Ar:O₂), and (c) Ar:O₂ to both sides (Ar:O₂/Ar:O₂). Results indicated that dry matter and current photosynthate (¹⁴C) were selectively partitioned to nodules and roots where N₂ was available. Both root and nodule growth on the Air side of Air/Ar:O₂ plants was significantly greater than the Ar:O₂ side. The relative partitioning of carbon and current photosynthate between roots and nodules on a half-root system was also affected by N₂ availability. The Ar:O₂ sides partitioned relatively more current photosynthate to roots (57%) than nodules (43%), while N₂-fixing root systems partitioned 36 and 64% of the carbon to roots and nodules, respectively. The Ar:O₂ atmosphere decreased root and nodule respiration by 80% and nitrogenase activity by 85% compared to half-root systems in Air while specific nitrogenase activity of nodules in Ar:O₂ was 50% of nodules supplied Air. Results indicated that nitrogen assimilation, whether from N₂ fixation or inorganic sources, had a localized effect on root development. Nodule development accounted for the major decrease in total photosynthate partitioning to non-N₂-fixing nodules. Soybean compensates for ineffective nodulation by controlling the flux of carbon to ineffective nodules and their associated roots.     

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.     

Nodulation of native woody legumes in Hong Kong, China

Woody legumes can play an important role in forest restoration on degraded land but the knowledge of woody legumes has lagged behind their uses. This study is a pioneer investigation to explore the ability of native woody legumes to form root nodules and fix nitrogen in Hong Kong. Nine sites of different habitat types were surveyed during both wet and dry seasons for two years. Young plants of woody legumes along studied transects were excavated. The patterns of nodulation and nodule morphology were recorded and the nitrogen fixing ability was tested by acetylene-reduction-assay. Twenty-eight species in 16 genera were examined, of which 20 species were nodulating and eight non-nodulating, including all six species in the Caesalpinioideae. Five species were new records to the world’s nodulation inventory. Bowringia callicarpa was a new species and genus examined, which was non-nodulating. The overall nodulation pattern was consistent with previous studies. Nodulation was more profuse in some shrub species while inconsistent in most tree species. Species with higher proportion of nodulated individual plants also tended to have more nodules in each plant. Spherical nodules were common in shrub and woody climber species whilst tree species usually had woody indeterminate nodules. Seasonal difference in the amount of senescent nodules was noted in most species. All the nodules tested by acetylene-reduction-assay were effectively nitrogen-fixing, with nitrogenase activity ranging from 4 μmol C₂H₄ g^?1 h^?1 to 20 μmol C₂H₄ g^?1 h^?1, which was comparable to other tropical tree species. The findings in nodulation pattern and nitrogen fixing ability of these species are essential in their application in forest restoration on degraded lands.     

Nitrogenase activity and N2 fixation are stimulated by elevated CO2 in a tropical N2-fixing tree

 Seeds of Gliricidia sepium, a fast-growing woody legume native to seasonal tropical forests of Central America, were inoculated with N₂-fixing Rhizobium bacteria and grown in environmentally controlled glasshouses for 67–71 days under ambient CO₂ (35 Pa) and elevated CO₂ (70 Pa) conditions. Seedlings were watered with an N-free, but otherwise complete, nutrient solution such that bacterial N₂ fixation was the only source of N available to the plant. The primary objective of our study was to quantify the effect of CO₂ enrichment on the kinetics of photosynthate transport to nodules and determine its subsequent effect on N₂ fixation. Photosynthetic rates and carbon storage in leaves were higher in elevated CO₂ plants indicating that more carbon was available for transport to nodules. A ¹⁴CO₂ pulse-chase experiment demonstrated that photosynthetically fixed carbon was supplied by leaves to nodules at a faster rate when plants were grown in elevated CO₂. Greater rates of carbon supply to nodules did not affect nodule mass per plant, but did increase specific nitrogenase activity (SNA) and total nitrogenase activity (TNA) resulting in greater N₂ fixation. In fact, a 23% increase in the rate of carbon supplied to nodules coincided with a 23% increase in SNA for plants grown in elevated CO₂, suggesting a direct correlation between carbon supply and nitrogenase activity. The improvement in plant N status produced much larger plants when grown in elevated CO₂. These results suggest that Gliricidia, and possibly other N₂-fixing trees, may show an early and positive growth response to elevated CO₂, even in severely N-deficient soils, due to increased nitrogenase activity. Received: 27 February 1996 / Accepted: 19 June 1996     

Spatial relationships between uninfected and infected cells in root nodules of soybean

In soybean (Glycine max (L.) Merr.) the uninfected cells of the root nodule are responsible for the final steps in ureide production from recently fixed nitrogen. Stereological methods and an original quantitative method were used to investigate the organization of these cells and their spatial relationships to infected cells in the central region of nodules of soybean inoculated with Rhizobium japonicum strain USDA 3I1B110 and grown with and without nitrogen (as nitrate) in the nutrient medium. The volume occupied by the uninfected tissue was 21% of the total volume of the central infected region for nodules of plants grown without nitrate, and 31% for nodules of plants grown with nitrate. Despite their low relative volume, the uninfected cells outnumbered the much larger infected cells in nodules of plants grown both without and with nitrate. The surface density of the interface between the ininfected and infected tissue in the infected region was similar for nodules in both cases also, the total range being from 24 to 26 mm²/mm³. In nodules of plants grown without nitrate, all sampled infected cells were found to be in contact with at least one uninfected cell. The study demonstrates that although the uninfected tissue in soybean nodules occupies a relatively small volume, it is organized so as to produce a large surface area for interaction with the infected tissue.     

Action of 2,4-DB and dalapon on the symbiotic properties ofLotus corniculatus (birdsfoot trefoil)

Summary Field trials carried out in 1965 and 1966 showed that 2,4-DB, alone or in combination with dalapon, reduced nodulation and tended to decrease the efficiency of nitrogen fixation in birdsfoot trefoil. Dalapon appeared to enhance the inhibitory action of 2,4-DB on nodulation. No obvious cytological differences could be detected in the nodules or in the isolated bacteroids of field-treated and untreated plants. Under growth chamber conditions, 2,4-DB drastically reduced trefoil growth and nodulation particularly in treatments where the herbicide came directly in contact with the plants. It appears that the reduction in nodulation and nitrogen fixation is a result of plant damage and abnormal root growth caused by 2,4-DB application.Autoradiographs indicated that the translocation of the herbicide was rapid, with detectable concentrations observed in young leaves, leafveins, roots, and nodules 12 hours after leaf-feeding of 2,4-DB-1-C¹⁴. The radio-activity appeared to accumulate with time (up to 5 days) in the growing root tips and nodules. Fractionation of excised nodules from trefoil plants demonstrated the presence of radioactivity in the cell debris, bacteroids, 29,000g pellet, plant ribosomes, and the soluble portion. The greatest accumulation of radioactivity occurred in the soluble fraction.The degradation of 2,4-DB and 2,4-D in trefoil was demonstrated by the evolution of C¹⁴O₂ from non-nodulated and aseptically growing plants leaf-fed with 2,4-DB-1-C¹⁴ or 2,4-D-1-C¹⁴.4-(2,4-dichlorophenoxy) butyric acid.2,2 dichloropropionic acid.     

The Effect of Potassium on the Fixation of Molecular Nitrogen by Root Nodules of Vicia faba

The effect of potassium supply of Vicia faba on the fixation of molecular nitrogen by root nodules was studied by using ¹⁵N-labeled molecular nitrogen. Plants well supplied with potassium showed higher contents of ¹⁵N in the soluble amino fraction and in the protein fraction of various plant organs as compared with plants of a lower potassium status. This effect was evident particularly in the root nodules. Assimilation experiments, carried out with ¹⁴CO₂, revealed that the content of radioactivity in the sugars and amino acids of the root nodules was increased by the potassium supply of the host plants. In particular, the content of ¹⁴C amino acids in the root nodules was influenced beneficially by potassium, which means that potassium favored the provision of reduced nitrogen (NH₃). It is postulated that the better carbohydrate supply of nodules, by plants well supplied with potassium, results in a higher carbohydrate turnover in the nodules and thus the provision of ATP and reducing electrons required by the nitrogenase is enhanced.     

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.     

Nitrogen Fixation in Peanut Nodules during Dark Periods and Detopped Conditions with Special Reference to Lipid Bodies

The peanut plant (Arachis hypogaea L.), unlike other known legumes, can sustain nitrogen fixation when prolonged periods of darkness or detopping curtail the supply of photosynthate to the nodule. This ability to withstand photosynthate stress is attributed to the presence of lipid bodies in infected nodule cells. In both dark-treated and detopped plants, the lipid bodies show a gradual decrease in numbers, suggesting their utilization as a source of energy and carbon for nitrogen fixation. Lipolytic activity can be localized in the lipid bodies, and the existence of β-oxidation pathway and, glyoxylate cycle is shown by the release of ¹⁴CO₂ from ¹⁴C lineoleoyl coenzyme A by the nodule homogenate.     

Growth of Sesbania rostrata (Brem) with stem nodules under controlled conditions

A series of experiments was carried out in an attempt to produce nodulated plants of Sesbania rostrata with qualities more closely resembling those in the wild than has been achieved to date. When groups of five plants were grown in a controlled climate chamber in pipes containing ～12dm³ modified Jensen's medium with 6mol m^?3 nitrate, the daily growth in height reached 5 cm and at 30 d the plants were ～40cm high. At this time, the stems were inoculated with Azorhizobium caulinodans ORS 571 and the medium replaced with Jensen's medium without nitrate. In the subsequent 19-d period ～300 nodules (representing >50% of the potential infection sites) developed on each stem. The nodules increased linearly in size over this time to ～15mg. Specific acetylene reduction activity, ARA ((μmol C₂H₄ mg^?1 h^?1) rose to 45 between days 5 and 10 after inoculation and plateaued; total ARA rose to ～200 μmol C₂H₄ plant^?1 h^?1. Under the conditions described the plants grew vigorously, and reproducibly uniform yields of nodules with high ARA activities were obtained. As outlined, the procedure offers a standard system in which, within a 2-week period after inoculation, individual strains of bacteria can be quantitatively compared in their ability to induce nodulation and N₂-fixation. Physiological and biochemical aspects of the nodulated system can be much more readily approached than with plants producing only root nodules. The inhibitory effects of stem nodules induced by wild type and two mutant strains of Azorhizobium on the development and activity of root nodules are described.     

Can a limitation in pholem supply to nodules account for the inhibitory effect of nitrate on nitrogenase activity in soybean?

Within 48 h of exposure of nodulated soybean [Glycine max (L.) Merr. cv. Harosoy 63 x Bradyrhizobium japonicum USDA 16] to 10 mM NO₃, significant decreases were observed in nodule-specific nitrogenase (EC 1.7.99.2) activity and CO₂ evolution and in the proportion of [¹⁴C]-labeled photosynthate partitioned to nodule biomass and respiration. These trends continued over the subsequent 3 days of the study period. Concomitant with these events was an 137% increase in the relative growth rate of the whole plant and a cessation in nodule growth. Although the concentration of total soluble sugar in nodules was not affected by NO₃ treatment, the concentration of starch declined to 13% of the control level after 2 days exposure to NO₃^?. In contrast to the effects of NO₃^?, nodules in which nitrogenase activity was partially inhibited by a 30 min exposure to 100% O₂, showed a 52% increase over control in the starch pool over a 72 h period. The results were compared with recent studies of NO₃^? inhibition of nitrogenase activity in legumes, and in contrast to these studies it was concluded that the inhibitory effects of NO₃^? could be accounted for by alterations in photosynthate partitioning to nodules. A hypothesis is proposed which attempts to account for the recent observation (J. K. Vessey, K. B. Walsh, and D. B. Layzell 1988. Physiol. Plant. 73: 113–121) that nitrogenase activity in phloem-limited and nitrate-inhibited nodules is limited by O₂ diffusion. This hypothesis separates the concepts of photosynthate partitioning and phloem supply from that of carbohydrate deprivation and related effects on the size of the carbohydrate pools in nodules.     

The physiological significance of the leaf nodules of psychotria

Summary In 6-month growth experiments it was found that leaf-nodulatedPsychotria mucronata seedlings grown in N-poor soil showed a restricted growth and developed severe nitrogen-deficiency symptoms in the leaves. Plants in the same soil supplied with NO₃-N showed healthy growth and dark green leaves. Detached Psychotria leaves bearing leaf nodules exposed to an atmosphere containing N¹⁵-labelled nitrogen gas or acetylene gas gave no evidence of nitrogen fixation, either in the light or in the dark or in both in succession. Therefore nitrogen fixation is probably not associated with the leaf nodules. Chlorophyll retention was observed around the leaf nodules in senescent Psychotria leaves. Psychotria leaf-nodule discs placed on oat leaves cause chlorophyll retention in the oat leaves below the discs. As chlorophyll retention is a common bioassay for cytokinins, these results indicate that a cytokinin-like substance is involved. With the aid of autoradiography and C¹⁴-labelled α-amino-isobutyric acid it was shown that this amino acid accumulates in the leaf nodules. Such directed transport is also a property of cytokinin.     

Hydrogen Recycling by Rhizobium leguminosarum Isolates and Growth and Nitrogen Contents of Pea Plants (Pisum sativum L.)

The ability to recycle H₂ evolved by nitrogenase is thought to be of importance in increasing the efficiency of N₂ fixation and to be a factor in increasing plant yield in symbiotic systems. To determine whether this ability is a significant factor in the Rhizobium leguminosarum-Pisum sativum L. system, plants were inoculated with R. leguminosarum isolates which differed in their ability to oxidize H₂ and in their relative efficiency of N₂ fixation. These plants were grown at three levels of irradiance and harvested after 3, 4, and 5 weeks of growth for determination of C₂H₂ reduction, H₂ evolution and uptake, plant dry weight, and N content. Plants inoculated with uptake hydrogenase-positive (Hup⁺) isolates did not exhibit higher dry weight or N content than those inoculated with Hup⁻ isolates under any of the growth conditions studied. The efficiency of the nitrogenase system of Hup⁻ isolates increased at a low irradiance, a factor which may allow them to compete successfully with Hup⁺ isolates. In some Hup⁺R. leguminosarum isolates, H₂ oxidation is coupled to ATP formation, whereas in others, it is not. There were no differences in plant dry weight and N content in plants inoculated with the two types and grown for 5 weeks at three irradiance levels. The addition of H₂ to Hup⁺ nodules whose supply of photosynthate had been removed by stem excision did not increase C₂H₂ reduction in either coupled or uncoupled types.     

Nodule protein synthesis and nitrogenase activity of soybeans exposed to fixed nitrogen

Nitrate or ammonium was added to soybean (Glycine max L. Merrill cv Corsoy) plants grown in plastic pouches 10 days after nodules first appeared. By the third day of treatment with 10 millimolar nitrate, nitrogenase specific activity (per unit nodule weight) had decreased to 15% to 25% of that of untreated plants. Longer incubations and higher concentrations of nitrate had no greater effect. In addition, exogenous nitrate or ammonium resulted in slower nodule growth and decreased total protein synthesis in both the bacterial and the plant portion of the nodule (as measured by incorporation of ³⁵S). Two-dimensional gel electrophoresis revealed that the nitrogenase components were not repressed or degraded relative to other bacteroid proteins. In the presence of an optimal carbon source, the nitrogenase specific activity of nodules detached from nitrate-treated plants was equivalent to that of nodules from untreated plants. These results are consistent with models that propose decreased availability or utilization of photosynthate in root nodules when legumes are exposed to fixed nitrogen.     

Interactions between Symbiotic Nitrogen Fixation, Combined-N Application, and Photosynthesis in Pisum sativum

The effect of photosynthetic photon flux density (PPFD) on nitrogen utilization was determined in peas (Pisum sativum L. cv. Alaska) inoculated with Rhizobium leguminosarum and treated with nutrient solutions containing no combined nitrogen, 16 mM NO₃^?, or 16 mM NH₄⁺. Plants were grown under controlled conditions at three PPFD values ranging from severely limiting to nearly saturating. Carboxylation efficiencies and CO₂-exchange rates were highest in the N₂-fixing plants and lowest in plants supplied with NH₄⁺, and they generally increased with increasing PPFD. Photoefficiencies increased with PPFD but did not differ appreciably with the form of nitrogen applied. Nitrogen fixation, calculated from C₂H₂-reduction and H₂-evolution data, was inhibited more by NH₄⁺ than by NO₃^?application. Inhibition was counteracted by increasing PPFD. Percentage nitrogen decreased with increasing PPFD in plants treated with combined nitrogen and increased in the plants dependent on N₂ fixation. The data reveal that photosynthetic efficiency and the capacity to fix N₂ in peas are functions of PPFD and the availability of combined nitrogen and that these two factors are interrelated.     

Developmental regulation of photosynthate distribution in leaves of rice

mRNA expression patterns of genes for metabolic key enzymes sucrose phosphate synthase (SPS), phosphoenolpyruvate carboxylase (PEPC), pyruvate kinase, ribulose 1,5-bisphosphate carboxylase/oxygenase, glutamine synthetase 1, and glutamine synthetase 2 were investigated in leaves of rice plants grown at two nitrogen (N) supplies (N_0.5, N_3.0). The relative gene expression patterns were similar in all leaves except for 9^th leaf, in which mRNA levels were generally depressed. Though increased N supply prolonged the expression period of each mRNA, it did not affect the relative expression intensity of any mRNA in a given leaf. SPS V_max, SPS limiting and PEPC activities, and carbon flow were examined. The ratio between PEPC activity and SPS V_max was higher in leaves developed at the vegetative growth stage (vegetative leaves: 5^th and 7^th leaves) than in leaves developed after the ear primordia formation stage (reproductive leaves: 9^th and flag leaves). PEPC activity and SPS V_max decreased with declining leaf N content. After using ¹⁴CO₂ the ¹⁴C photosynthate distribution in the amino acid fraction was higher in vegetative than in reproductive leaves when compared for the same leaf N status. Thus, at high PEPC/SPS activities ratio, more ¹⁴C photosynthate was distributed to the amino acid pool, whereas at higher SPS activity more ¹⁴C was channelled into the saccharide fraction. Thus, leaf ontogeny was an important factor controlling photosynthate distribution to the N- or C-pool, respectively, regardless of the leaf N status.     

Effect of host shoot clipping on carbon and nitrogen sources for arbuscular mycorrhizal fungi

The effect of clipping of the host-plant shoot on the sources of carbon and nitrogen for the arbuscular mycorrhizal (AM) fungus Gigaspora margarita was determined by measuring ¹³C in spores and hyphae in cocultures of C₃ and C₄ plants and by differential ¹⁵N labeling. C₃ and C₄ plants, which have different δ¹³C values, were grown in the same container separated by a series of hyphal compartments. The C₃ and C₄ plants were applied with ¹⁴N- and ¹⁵N-urea, respectively. After clipping of the C₃ shoots, spore δ ¹³C gradually approached that of the C₄ roots. Hyphal δ ¹³C paralleled that of spores. Spore % ¹⁵N was similar to that of mineral N in the C₄ plant compartment. Thus C in G. margarita coming from the clipped plants decreased with time. This demonstrates that C in AM fungi comes from living plants, whilst the N in spores comes mostly from the soil. Accepted: 28 November 2000     

Effect of changes in shoot carbon-exchange rate on soybean root nodule activity

The effect of short- and long-term changes in shoot carbon-exchange rate (CER) on soybean (Glycine max [L.] Merr.) root nodule activity was assessed to determine whether increases in photosynthate production produce a direct enhancement of symbiotic N₂ fixation. Shoot CER, root + nodule respiration, and apparent N₂ fixation (acetylene reduction) were measured on intact soybean plants grown at 700 microeinsteins per meter per second, with constant root temperature and a 14/10-hour light/dark cycle. There was no diurnal variation of root + nodule respiration or apparent N₂ fixation in plants assayed weekly from 14 to 43 days after planting. However, if plants remained in darkness following their normal dark period, a significant decline in apparent N₂ fixation was measured within 4 hours, and decreasing CO₂ concentration from 320 to 90 microliters CO₂ per liter produced diurnal changes in root nodule activity. Increasing shoot CER by 87, 84, and 76% in 2-, 3-, and 4-week-old plants, respectively, by raising the CO₂ concentration around the shoot from 320 to 1,000 microliters CO₂ per liter, had no effect on root + nodule respiration or acetylene-reduction rates during the first 10 hours of the increased CER treatment. When the CO₂-enrichment treatment was extended in 3-week-old plants, the only measured parameter that differed significantly after 3 days was shoot CER. After 5 days of continuous CO₂ enrichment, root + nodule respiration and acetylene reduction increased, but such changes reflected an increase in root nodule mass rather than greater specific root nodule activity. The results show that on a 24-hour basis the process of symbiotic N₂ fixation in soybean plants grown under controlled environmental conditions functioned at maximum capacity and was not limited by shoot CER. Whether N₂-fixation capacity was limited by photosynthate movement to root nodules or by saturation of metabolic processes in root nodules is not known.