Lupin Nodules Research Articles

Glutamate dehydrogenase of lupin nodules: Kinetics of the deamination reaction

The reaction of glutamate dehydrogenase ( l-glutamate: NAD + oxidoreductase (deaminating) EC 1.4.1.2) from lupin nodules has been investigated in the direction of deamination by means of steady state velocity studies in the absence of products and inhibition studies with products and substrate analogs. The results are qualitatively and quantitatively consistent with a fully ordered reaction mechanism in which NAD + binds to the enzyme first followed by l-glutamate. The order of product release is proposed to be NH 4 + followed by 2-oxoglutarate and then NADH. In addition, product inhibition data indicate the formation of an enzyme-NAD-oxoglutarate dead-end complex.

Effects of initial watering on nodulation of lupins

The need for free water movement through the soil after sowing to obtain good nodulation of yellow lupins (Lupinus luteus) was verified in a glasshouse experiment. Withholding overhead watering after sowing reduced the number of plants nodulated by approximately 30% when compared with surface watering at either one or 3 days after sowing. The amount of nodule tissue of nodulated plants was also lowered. Lime pelleting or a higher rate of inoculation increased the number of plants nodulated, but did not increase the amount of nodule tissue on nodulated plants. With watering 3 days after sowing, fewer plants nodulated and they had less nodule tissue than with watering after one day, unless lime-pelleted seed was used or (in the case of number of plants nodulated) a higher rate of inoculum was used.

Wirksamkeit der symbiontischen N2-Fixierung der Körnerleguminosen in Abhängigkeit von Rhizobienimpfung, Substrat, N-Düngung und 14C-saccharoselieferung

Cultivation experiments (Mitscherlich-vessels, quartz sand, 15N-labelled soil, 15N-fertilizer) showed, that various strains of Rhizobium lupini (white and yellow lupines) and of Rhizobium leguminosarum (field beans and peas) induced a different N2-fixation of the inoculated plants, the most effective Rhizobium strains being 367a, Cz, T3, 271 (Rh. lupini), and Azotogen (Rh, leguminosarum). Yellow lupines and field bean plants were supplied with N2 from the air considerably better than white lupines and peas after inoculation with the most effective Rhizobium strains. Application of mineral N to the white lupines and peas not only substituted the inhibited N2-fixation, but increased N amounts in the plants. White lupines fixed more N2 under soil conditions than in quartz sand. An experiment with steam-sterilized and 15-labelled soil as a comparative substrate showed, that this finding was mainly caused by an additional Rhizobium infection from the soil. Contrary to field beans and yellow lupines which fix N2 up to ripeness, white lupines and peas finished N2-fixation in the time of flowering. Mineral-N applied at that time was an additional source of N for last-named plants and they utilized it for production of higher protein yields. Continual spraying of white lupine plants with 14C-labelled sucrose solution after the time of flowering caused continuance of N2-fixation up to the stage of ripeness. It is assumed that the cause of this effect was the competition of growing seeds and nodules for the photosynthates. The supply of nodules was inadequate without external sucrose application. Mineral N inhibited the sucrose-induced N2-fixation of white lupine nodules and their consumption of photosynthates. Consequently, the applied 14C was transported into seeds to a larger extent. The investigations allow the following conclusion: Effective N2-fixation requires nodules being a powerful sink for assimilates on the basis of a highly efficient photosynthetic system of the host plant.

Effects of Protein Synthesis Inhibitors on Acetylene Reduction Activity of Lupin Root Nodules

Rifampicin and D-threo-chloramphenicol inhibited the incorporation of [35S]methionine into purified bacteroid suspensions, and into the bacteroid fraction but not the plant cytoplasmic fraction of cultured nodules. Cycloheximide and anisomycin inhibited [35S]methionine incorporation into the plant cytoplasmic fraction of cultured nodules; at early times they inhibited incorporation into the bacteroid fraction, but at later times this effect was reversed. Chloramphenicol, rifampicin, spectinomycin, cycloheximide and anisomycin all prevented the induction of acetylene reduction activity in immature nodules; spectinomycin did not prevent induction in nodules containing a spectinomycin-resistant Rhizobium. Neither rifampicin nor chloramphenicol inhibited the acetylene reduction activity of mature nodules, but cycloheximide and anisomycin caused rapid loss of activity. Cycloheximide did not inhibit the acetylene reduction activity of Rhizobium strain 32H1 in pure cultures. The results suggest that both plant cytoplasmic protein synthesis and bacteroid protein synthesis are needed for the induction of nitrogenase activity in developing lupin nodules, and that plant cytoplasmic protein synthesis but not bacteroid protein synthesis is needed for the maintenance of nitrogenase activity at high levels.

Stereospecificity and NADH—H 2O hydrogen exchange of NADH-dependent glutamate synthase from lupin nodules

Stereospecificity and NADH—H 2O hydrogen exchange of NADH-dependent glutamate synthase from lupin nodules

Kinetic Mechanism of NADH‐Dependent Glutamate Synthase from Lupin Nodules

From initial-rate studies, a partially random kinetic mechanism has been deduced for NADH-dependent glutamate synthase from lupin nodules. The mechanism involves compulsory binding of NADH as first substrate, followed by random-order binding of glutamine and 2-oxoglutarate. Patterns of inhibition by glutamate substantiate the mechanism. Dithionite was incapable of acting as an alternative reducing substrate although it is known to reduce the flavine groups of the enzyme. The implications of these results are discussed. Published rate equations for this type of mechanism were found to be unsatisfactory for this enzyme and suitable new equations are produced. These equations should have general application where the obligatory first substrate binds very tightly.

The involvement of aspartate aminotransferases in ammonium assimilation in lupin nodules

Aspartate aminotransferase (AAT) activity has been detected in the plant and bacteroid fractions of lupin nodules, and in free-living Rhizobium lupini. Two electrophoretically distinct forms of AAT were detected in the plant fraction of the nodule and a third form in the bacteroid fraction. AAT activity increased in the plant fraction during nodule development and this increase may be due to an increase in the activity of one of the AAT forms in this fraction. The single form of AAT detected in the bacteroid fraction had the same electrophoretic mobility as that detected in free-living R. lupini. The nodulated roots of lupins, grown in a media supplemented with nitrate and ammonium, had a 3- and 4-fold lower activity of AAT and nitrogenase activity respectively, compared to the nodulated roots of plants grown in the absence of added nitrogen. A role for the plant AAT in ammonium assimilation in lupin nodules is proposed.

Polypeptide synthesis by Rhizobium bacteroids and bacteria

When Rhizobium bacteroids (strain NZP 2257) from lupin nodules were isolated and incubated aerobically at high osmolarity, they incorporated [ 35S]methionine into a characteristic set of polypeptides; many of these polypeptides coelectrophoresed on SDS-polyacrylamide gels with the bacteroid polypeptide bands stained by Coomassie blue. The labelled polypeptides were stable for several hours in pulse-chase experiments. Changes in the concentration of H +, K + and Mg 2+ in the incubation mixture affected overall incorporation of label, but not the relative incorporation into different polypeptides. A similar set of bacteroid polypeptides was labelled in situ when detached nodules were fed [ 35S]methionine. Distinctive labelling patterns were observed with bacteroid suspensions from mature and immature nodules, with a transitional pattern at the time when nitrogenase activity appeared. Two of the major labelled components in mature bacteroids had estimated molecular weights of 60- and 34-kilodaltons similar to values reported by others for the constituent polypeptides of nitrogenase. Bacteroids of the same Rhizobium strain grown in different plant hosts gave similar polypeptide labelling patterns in purified suspensions, but bacteroids of different Rhizobium strains gave different patterns. The polypeptide labelling patterns obtained using broth-cultured Rhizobium bacteria from various growth stages and growth media differed from those obtained using bacteroids of the same strain.

Carbon Dioxide Fixation by Lupin Root Nodules

Labeling studies using detached lupin (Lupinus angustifolius) nodules showed that over times of less than 3 minutes, label from [3,4-(14)C]glucose was incorporated into amino acids, predominantly aspartic acid, to a much greater extent than into organic acids. Only a slight preferential incorporation was observed with [1-(14)C]- and [6-(14)C]glucose, while with [U-(14)C]-glucose more label was incorporated into organic acids than into amino acids at all labeling times. These results are consistent with a scheme whereby the "carbon skeletons" for amino acid synthesis are provided by the phosphoenolpyruvate carboxylase reaction.A comparison of (14)CO(2) release from nodules supplied with [1-(14)C]- and [6-(14)C]glucose indicated that the oxidative pentose phosphate pathway accounted for less than 6% of glucose metabolism. Several enzymes of the oxidative pentose phosphate and glycolytic pathways were assayed in vitro using the 12,000g supernatant fraction from nodule homogenates. In all cases, the specific activities were adequate to account for the calculated in vivo fluxes.Three out of four diverse treatments that inhibited nodule nitrogen fixation also inhibited nodule CO(2) fixation, and in the case of the fourth treatment, replacement of N(2) with He, it was shown that the normal entry of label from exogenous (14)CO(2) into the nodule amino acid pool was strongly inhibited.

Glutamate dehydrogenase of lupin nodules: Purification and properties

Glutamate dehydrogenase, GDH ( l-glutamate: NAD + oxidoreductase (deaminating) EC 1.4.1.2) was purified from the plant fraction of lupin nodules and the purity of the preparation established by gel electrophoresis and electrofocusing. The purified enzyme existed as 4 charge isozymes with a MW of 270000. The subunit MW, as determined by dodecyl sulphate electrophoresis, was 45 000. On the basis of the results of the MW determinations a hexameric structure is proposed for lupin-nodule GDH. The pH optima for the enzyme were pH 8.2 for the amination reaction and pH 8.8 for the deamination reaction. GDH from lupin nodules showed a marked preference for NADH over NADPH in the amination reaction and used only NAD + for the deamination reaction. Pyridoxal-5′-P and EDTA inhibited activity. The enzyme displayed Michaelis-Menten kinetics with respect to all substrates except NAD +. When NAD + was the varied substrate, there was a deviation from Michaelis-Menten behaviour towards higher activity at high concentrations of NAD +.

Studies on hydrogenases in legume root nodule bacteroids: Effect of ATP.

Hydrogenase activity (Tritium uptake activity) in cell-free extracts of bacteroids from soybean and lupin nodules was separated into two parts, whose activities were different from each other in responsiveness to ATP, by differential centrifugation, Sephadex gel filtration or DEAE-cellulose column chromatography. One was stimulated and the other was either inhibited or not affected by ATP.

The DNA content of Rhizobium (strain NZP 2257) bacteroids and bacteria

The DNA content of Rhizobium (strain NZP 2257) bacteroids and bacteria was measured by cytofluorometry of individual cells and by chemical estimates on cell populations. Both methods showed that bacteroids from lupin nodules have a higher average DNA content than bacteria, and that bacteroid DNA content increases with nodule age. The DNA content of bacteroids showed a positive correlation with their sedimentation rate in sucrose gradients, but changes in the sedimentation rate of broth-cultured bacteria induced by adding mannitol to the growth medium were not associated with any alteration in the DNA content of stationary phase cells.

Membranes in lupin root nodules. II. Preparation and properties of peribacteroid membranes and bacteroid envelope inner membranes from developing lupin nodules.

Peribacteroid membranes and bacteroid envelope inner membranes have been isolated from developing lupin nodules. Isolation of the peribacteroid membranes was achieved by first preparing membrane-enclosed bacteroids free from other plant organelles or membranes. The peribacteroid membranes were then released by osmotic shock and purified by centrifugation to equilibrium on sucrose gradients. The bacteroids were broken in a pressure cell and the bacteroid envelope inner membranes were isolated using sucrose gradient fractionation of the bacteroid total envelope preparation. The density of the peribacteroid membranes decreased during the period of development of N2-fixation in lupin nodules from 1.148 g/ml for nodules from 12-day plants to 1.137 g/ml for nodules from 18-day plants. The density of the bacteroid envelope inner membranes from nodules from 18-day plants was 1-153 g/ml. The identity and homogeneity of the isolated membranes was established, by comparison with membranes in intact nodules, using phosphotungstic acid and silver staining of thin sections and particle densitites on faces of freeze-fracture replicas of the membranes. Analyses for NADH oxidase and succinate dehydrogenase, spectral analyses and gel-electrophoretic analysis of proteins were also used to characterize the membrane and soluble protein fractions from the nodules. The ratio of lipid to protein was 6.1 for the peribacteroid membranes and 2.5 for the bacteroid envelope inner membranes. Leghaemoglobin was localized in the plant cytoplasm in lupin nodules and not in the peribacteroid space.

Membranes in lupin root nodules. I. The role of Golgi bodies in the biogenesis of infection threads and peribacteroid membranes.

The process of infection of lupin nodule cells by rhizobia was examined using thin-section and freeze-fracture electron-microscopic techniques to characterize the properties of different membranes and to establish relationships between them. The membranes of the Golgi bodies and the endoplasmic reticulum stained with zinc iodide-osmium tetroxide but not with phosphotungstic acid or silver. By contrast the infection thread membranes, peribacteroid membranes, plasma membranes and membranes of cytoplasmic vesicles did not stain with zinc iodide-osmium tetroxide but stained with phosphotungstic acid and silver. The peribacteroid membranes and plasma membranes are, however, different from each other since the particle density on the E face of freeze-fracture replicas of plasma membranes was twice that on the E face of the peribacteroid membranes. An examination of the tips of the infection threads in the cytoplasm of the plant cells, showed that the rhizobia bud off from the infection threads enclosed in the infection thread membranes. The rhizobia continue to divide still surrounded by membranes of plant origin, namely the peribacteroid membranes. Cytoplasmic vesicles are observed in both thin-section and freeze-fracture preparations of nodule tissue closely associated with, and apparently produced by, Golgi bodies. Formation of the walls and membranes of the infection threads and of the peribacteroid membranes involves fusion of the cytoplasmic vesicles with these membranes. It is proposed that the process of infection of plant cells in lupin nodules involves a change in the function of the Golgi body system for the biogenesis of plant cell walls and plasma membranes to include the synthesis of the walls and membranes of the infection threads and also the peribacteroid membranes.

Preparation and Fractionation of Rhizobium Bacteroids by Zone Sedimentation through Sucrose Gradients

Zone sedimentation through sucrose gradients was used for preparing Rhizobium bacteroids from lupin nodules and for separating them into slowly and rapidly sedimenting fractions.The purity of the bacteroids was established by electron microscopy and by enzyme assays, and they were shown to contain a CO-insensitive cytochrome c oxidase.Bacteroids sedimented more rapidly than broth-cultured Rhizobium bacteria, and bacteroids from old nodules sedimented more rapidly than bacteroids from young nodules.THERE APPEAR TO BE AT LEAST TWO FORMS OF BACTEROID IN OLD NODULES: slowly sedimenting bacteroids with moderate colony-forming ability resembling the bacteroids found in young nodules, and rapidly sedimenting bacteroids with much lower colony-forming ability.

Enzymes of nitrogen metabolism in legume nodules. Purification and properties of NADH-dependent glutamate synthase from lupin nodules.

An NADH-dependent glutamate synthase has been purified 500-fold from the plant cytoplasm fraction of Lupinus angustifolius nodules. It consists of a single polypeptide chain, Mr 235000. The optimum pH is 8.5, at which Km values for 2-oxoglutarate, glutamine and NADH are 39 micrometer, 400 micrometer and 1.3 micrometer respectively. The catalytic centre activity is of the order of 70 s-1 and is independent of pH between 6.5 and 9.5. Glutamate synthase is inhibited by glutamic acid, oxaloacetic acid, aspartic acid and asparagine, all competitive with 2-oxoglutarate; and by NAD+, which is competitive with NADH. There is evidence of two flavine prosthetic groups per enzyme molecule.

Carbon Dioxide Fixation by Lupin Root Nodules

In vivo CO(2) fixation and in vitro phosphoenolpyruvate (PEP) carboxylase levels have been measured in lupin (Lupinus angustifolius L.) root nodules of various ages. Both activities were greater in nodule tissue than in either primary or secondary root tissue, and increased about 3-fold with the onset of N(2) fixation. PEP carboxylase activity was predominantly located in the bacteroid-containing zone of mature nodules, but purified bacteroids contained no activity. Partially purified PEP carboxylases from nodules, roots, and leaves were identical in a number of kinetic parameters. Both in vivo CO(2) fixation activity and in vitro PEP carboxylase activity were significantly correlated with nodule acetylene reduction activity during nodule development. The maximum rate of in vivo CO(2) fixation in mature nodules was 7.9 nmol hour(-1) mg fresh weight(-1), similar to rates of N(2) fixation and reported values for amino acid translocation.The results suggest that the oxaloacetete used as the primary "carbon skeleton" acceptor for ammonia assimilation and amino acid synthesis in lupin nodules is provided via the PEP carboxylase reaction rather than through the tricarboxylic acid cycle. The source of PEP is presumably glycolysis, while the major source of CO(2) is inferred to be respiration.

Changes in the Number, Viability, and Amino-acid-incorporating Activity of Rhizobium Bacteroids during Lupin Nodule Development

Between days 10 and 21 after inoculation of Lupinus angustifolius seedlings with Rhizobium NZP 2257, the average nodule fresh weight increased 3-fold and the number of bacteroids per nodule increased more than 10-fold.The viability of Rhizobium bacteroids, as judged by their ability to form colonies on yeast-extract agar, declined from about 10% on days 10 and 11 after inoculation to about 0.3% on days 14 to 25. Bacteroid viability was highly sensitive to osmolarity.At optimal pH and K and Mg ion concentrations, the incorporation of (14)C-glycine into isolated bacteroids was also very sensitive to osmolarity, and fell in parallel with bacteroid viability during nodule development.WE SUGGEST THAT AT LEAST TWO PROCESSES CONTRIBUTE TO BACTEROID NONVIABILITY: a reversible change in the cell wall structure occurring between days 10 and 14 after inoculation, and a subsequent irreversible change.

Synthesis and turnover of leghaemoglobin in lupin root nodules

1. The problem of whether leghaemoglobin is synthesized on plant or bacterial ribosomes in root nodules of yellow lupin has been examined. 2. Leghaemoglobin, soluble plant protein and soluble bacteroid protein were labelled with 14C administered by uptake of 14CO 2. 3. Exposure of roots to 1 mM d- threo-chloramphenical resulted in inhibition of soluble bacteroid protein synthesis, but leghaemoglobin synthesis and soluble plant protein synthesis were unaffected. This result is consistent with leghaemoglobin being synthesized on plant ribosomes. 4. After nitrogen-fixing plants had been supplied with a pulse of 14CO 2, the decay of specific radioactivity of nodule protein fractions was observed. Leghaemoglobin had an apparent half-life of 18 days and is a stable protein in nitrogen-fixing yellow lupin nodules.

Kinetics of acetylene and CN- reduction by the N 2-fixing system of Rhizobium Lupino

1. 1. N 2-fixing extracts have been successfully isolated and party purified from bacteroids of Rhizobium lupini obtained from yellow lupin nodules. These extracts catalysed acetylene and CN- reduction, demonstrating the same substrate versatility as nitrogenase from other organisms. 2. 2. The enzyme saturation curves by ATP for both acetylene and CN- reduction were closely similar. These indicate that nitrogenase from this source has several sites for binding ATP. Intermediary plateaus suggest that cooperation of these sites involves both positive and negative changes in either binding or catalytic activity as the enzyme is saturated with ATP. 3. 3. Curves for inhibition of acetylene and CN- reduction by ADP also displayed mixed positive and negative cooperativity. It is proposed that ADP has an inverse effect to ATP on the rate of reductive catalysis by nitrogenase. 4. 4. The complex kinetics displayed are consistent with a hypothesis that on aspect of the role of ATP is to induce conformational changes in nitrogenase. Also, the kinetics provide a possible mechanism for nitrogenase regulation by the available energy supply. 5. 5. Tests with a variety of nitrogenous products of fixation and other compounds failed to provide convincing evidence for feed-back inhibition of the nitrogenase in Rhizobium lupini.