Localizations of aluminum in soybean bacteroids and seeds.

Aluminum, long known to be detrimental to soybean productivity, was localized in the polyphosphate granules (PPG) of bacteroids in root nodules of soybean plants. By using energy-dispersive X-ray analysis, bacteroids in early infections were shown to have typical PPG constituents. However, in PPG in older infections and after the bacteroids were digested intracellularly, aluminum was also detected. These results indicate that aluminum accumulates in PPG after a period when organisms have been resident in host cells and that high levels of aluminum were present in the bacteroids at the time of their demise. At least some of the aluminum in these laboratory-grown plants could have come from the seeds used.     

Utilization of aldehydes and alcohols by soybean bacteroids

Aldehydes, alcohols and acids were tested for their ability to support acetylene reduction and oxygen consumption by Rhizobium japonicum bacteroids isolated from soybean nodules. Several alcohols and aldehydes increased acetylene reduction and oxygen uptake. This is consistent with the concept that the plant nodule cytosol can metabolize carbohydrate via anaerobic fermentative pathways.     

Nitrite inhibition of nitrogenase from soybean bacteroids

Nitrogenase from soybean bacteroids was purified and used to study NO ₂ ^– effects either as unfractionated enzyme or as reconstituted enzyme from separated nitrogenase components I and II. Partially purified enzyme was strongly inhibited by nitrite at concentrations less than 0.1 mM. This inhibition was typically referred to as competitive with an inhibition constant (K _i) for NO ₂ ^– which was 5.2 mM. Kinetics studies showed an abnormally low apparent constant of association between enzyme and NO ₂ ^– (k _a=60 M^-1·s^-1). Nitrite appeared to bind to the MoFe protein, without any effect on Fe component, giving a completely reversible inhibition. Nitrite was found not to be an alternative substrate for nitrogenase.Abbreviations TES N-tris (hydroxymethyl) methyl-2-aminoethane sulfonic acid - PPG Polypropylene glycol     

Spontaneous luminescence from soybean Rhizobium bacteroids

Abstract: A remarkable spontaneous photon emission was observed in isolated bacteroids of three strains of soybean rhizobia from different genera, but not for the same rhizobia when cultured in liquid medium. The photon emission is oxygen-dependent and can be inhibited by desferal or dipyridyl (both good iron-chelating agents), superoxide dismutase or β-carotene. It is enhanced by catalase. The emission spectrum indicates that singlet oxygen is partly responsible for the luminescence.     

Viability of Rhizobium bacteroids isolated from soybean nodule protoplasts

Bacteriods isolated from protoplasts taken from Rhizobium japonicum induced root nodule of Glycine max L. showed complete viability when plated onto a conventional rhizobial growth medium supplemented with 0.2 M Mannitol. The same medium but without extra mannitol resulted in the absence of colony formation. The protoplast isolation method eliminated the possibility of contaminant bacteria from infection threads to be scored. The redifferentiated bacteroid clones have the same genetical characteristics as the orginal inoculum strain. This and other recent findings of bacteroid viability are discussed in the light of the existing belief that bacteroids are non-viable.     

Characteristics of the H2 oxidizing system in soybean nodule bacteroids

A derivative of Rhizobium japonicum (strain 122 DES) has been isolated which forms nodules on soybeans that evolve little or no H₂ in air and efficiently fixes N₂. Bacteroids isolated from nodules formed by strain 122 DES took up H₂ with O₂ as the physiological acceptor and appeared to be typical of those R. japonicum strains that possess the H₂ uptake system. The hydrogenase system in soybean nodules is located within the bacteroids and activity in macerated bacteroids is concentrated in a particulate fraction. The pH optimum for the reaction is near 8.0 and apparent K _m values for H₂ and O₂ are 2 M and 1 M, respectively. The H₂ oxidizing activity of a suspension of 122 DES bacteroids was stable at 4°C for at least 4 weeks and was not particularly sensitive to O₂. Neither C₂H₂ nor CO inhibited O₂ dependent H₂ uptake activity.Non-physiological electron acceptors of positive oxidation reduction potential also supported H₂ uptake by bacteroids. The rate of H₂ uptake with phenazine methosulfate as the acceptor was greater than that with O₂. When methylene blue, triphenyltetrazolium, potassium ferricyanide or dichlorophenolindophenol were added to bacteriod suspensions, without preincubation, rates of H₂ uptake were supported that were lower than those in the presence of O₂. Preincubation of the bacteroids with acceptors increased the rates of H₂ uptake. No H₂ evolution was observed from reaction mixtures containing bacteroid suspensions and reduced methyl or benzyl viologens. Of a series of carbon substrates added to bacteroid suspensions only acetate, formate or succinate at concentrations of 50 mM resulted in 20% or greater inhibition of H₂ oxidation.The H₂ uptake capacity of isolated 122 DES bacteroids (expressed on a dry bacteroid basis) was at least 10-fold higher than the rate of the nitrogenase reaction in nodules expressed on a comparable basis. Since about 1 mol of H₂ is evolved for every mol of N₂ reduced during the N₂ fixation reaction, these observations explain why soybean nodules formed by strain 122 DES and other strains with high H₂ uptake activities have a capacity for recycling all the H₂ produced from the nitrogenase reaction.Abbreviations PMS PHenazine methosulfate - MB Methylene blue     

Soluble aldehyde dehydrogenase and metabolism of aldehydes by soybean bacteroids.

A soluble aldehyde dehydrogenase (EC 1.2.1.3) was partially purified from Rhizobium japonicum bacteroids and from free-living R. japonicum 61A76. The enzyme was activated by NAD+, NADH, and dithiothreitol, and it reduced NAD(P)+. Acetaldehyde, propionaldehyde, butyraldehyde, benzaldehyde, and succinic semialdehyde were substrates. The Km for straight-chain aldehydes decreased with increasing carbon chain length. The aldehyde dehydrogenase was inhibited by 6-cyanopurine, but not by metronidazole. These compounds inhibited acetylene reduction, but not respiration, by isolated bacteroids.     

Isolation and oxidative properties of mitochondria and bacteroids from soybean root nodules

Summary A method for the separation and purification of bacteroids and mitochondria from nodules of soybean roots is described. Cross contamination between these two oxidative fractions was easily assessible by using NADH oxidase and -hydroxybutyrate dehydrogenase respectively as specific mitochondrial and bacteroid markers. Bacteroid respiration was characterized by substantial endogenous respiration which could be reduced by keeping plants in the dark prior to isolation, and stimulated by uncoupler or organic acids. Nodule mitochondria readily oxidized external NADH and a range of tricarboxylic acid cycle intermediates, with good respiratory control. A major difference between nodule and root mitochondria was the former's high sensitivity to the inhibitors rotenone and cyanide. This indicates a reduced capacity for non-phosphorylating electron transport in nodule mitochondria, which may be related to the large energy demand during ammonia assimilation in nodule cells.     

Isolation of bacteria, transforming bacteria, and bacteroids from soybean nodules

Postnuclei supernatant of soybean (Glycine max cv. Chippewa 64) nodule homogenate was fractionated by stepwise sucrose density gradient centrifugation into supernatant, endoplasmic reticulum and mitochondria, and three distinct bands with 1.22, 1.25, and 1.27 g/cm³ of peak density. Based on their enzymic activities, composition of electron transport components, and ultrastructural characteristics, the lightest band appears to be the mature bacteroids; the intermediate band the transforming bacteria; and the heaviest, the bacteria. The isolation procedure separates nodule symbionts into different functional and developmental fractions, and it may be a valuable tool for studies involving development, regulation, and senescence of bacteroids in the nodule.     

Hydrogen inhibition of nitrogen reduction by nitrogenase in isolated soybean nodule bacteroids

Dihydrogen, a by-product of biological nitrogen fixation, is a competitive inhibitor of N₂ reduction by nitrogenase. To evaluate the significance of H₂ inhibition in vivo, we have measured the apparent inhibition constant for H₂ inhibition of N₂ reduction in Bradyrhizobium japonicum bacteroids isolated from soybean nodules. The rate of N₂ reduction was measured as ammonia production by bacteroids incubated in a buffer containing 200 micromolar leghemoglobin and 10 millimolar succinate under 0.02 atmosphere O₂ and various concentrations of N₂ and H₂. The apparent inhibition constant for H₂ under these conditions was determined to be approximately 0.03 atmosphere. This relatively low value strengthens the proposal that H₂ inhibition of N₂ reduction may be a significant factor in lowering the efficiency of nitrogen fixation in legume nodules.     

Isolation and characterization of the membrane envelope enclosing the bacteroids in soybean root nodules

The membrane envelope enclosing the bacteroids in soybean root nodules is shown by ultrastructural and biochemical studies to be derived from, and to retain the characteristics of, the host cell plasma membrane. During the early stages of the infection process, which occurs through an invagination, Rhizobium becomes surrounded by the host cell wall and plasma membrane, forming the infection thread. The cell wall of the infection thread is degraded by cellulolytic enzyme(s), leaving behind the enclosed plasma membrane, the membrane envelope. Cellulase activity in young nodules increases two- to threefold as compared to uninfected roots, and this activity is localized in the cell wall matrix of the infection threads. Membrane envelopes were isolated by first preparing bacteroids enclosed in the envelopes on a discontinuous sucrose gradient followed by passage through a hypodermic needle, which released the bacteroids from the membranes. This membrane then sedimented at the interface of 34--45% sucrose (mean density of 1.14 g/cm3). Membranes were characterized by phosphotungstic acid (PTA)-chromic acid staining. ATPase activity, and localization, sensitivity to nonionic detergent Nonidet P-40 (NP-40) and sodium dodecyl sulfate (SDS) gel electrophoresis. These analyses revealed a close similarity between plasma membrane and the membrane envelope. Incorporation of radioactive amino acids into the membrane envelope proteins was sensitive to cycloheximide, suggesting that the biosynthesis of these proteins is primarily under host-cell control. No immunoreactive material to leghemoglobin antibodies was found inside or associated with the isolated bacteroids enclosed in the membrane envelope, and its location is confined to the host cell cytoplasmic matrix.     

Utilization of nitrate by bacteroids of Bradyrhizobium japonicum in the soybean root nodule

Bacteroids of Bradyrhizobium japonicum strain CB1809, unlike CC705, do not have a high level of constitutive nitrate reductase (NR; EC 1.7.99.4) in the soybean (Glycine max. Merr.) nodule. Ex planta both strains have a high activity of NR when cultured on 5 mM nitrate at 2% O₂ (v/v). Nitrite reductase (NiR) was active in cultured cells of bradyrhizobia, but activity with succinate as electron donor was not detected in freshly-isolated bacteroids. A low activity was measured with reduced methyl viologen. When bacteroids of CC705 were incubated with nitrate there was a rapid production of nitrite which resulted in repression of NR. Subsequently when NiR was induced, nitrite was utilized and NR activity recovered. Nitrate reductase was induced in bacteroids of strain CB1809 when they were incubated in-vitro with nitrate or nitrite. Increase in NR activity was prevented by rifampicin (10 g· ml^-1) or chloramphenicol (50 g·ml^-1). Nitrite-reductase activity in bacteroids of strain CB1809 was induced in parallel with NR. When nitrate was supplied to soybeans nodulated with strain CC705, nitrite was detected in nodule extracts prepared in aqueous media and it accumulated during storage (1°C) and on further incubation at 25°C. Nitrite was not detected in nodule extracts prepared in ethanol. Thus nitrite accumulation in nodule tissue appears to occur only after maceration and although bacteroids of some strains of B. japonicum have a high level of a constitutive NR, they do not appear to reduce nitrate in the nodule because this anion does not gain access to the bacteroid zone. Soybeans nodulated with strains CC705 and CB1809 were equally sensitive to nitrate inhibition of N₂ fixation.Abbreviations NR nitrate reductase - NiR nitrite reductase - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Respiratory pathways in aged soybean seeds

Respiratory activities in soybean seeds (Glvcine max (L.) Merr. cv. Chippewa 64) have been examined in the first minutes after water imbibition and after three hours of imbibition, using either particles or intact axes. Cyanide and azide were utilized as inhibitors of the cytochrome oxidase pathway of respiration, and SHAM inhibitions were interpreted as effects on the alternative pathway, since in unaged axes inhibitions by SHAM were obtained only when respiratory activity had either been restricted with inhibitors of the cytochrome oxidase pathway or expanded by an uncoupler (CCCP). From the experiments with these inhibitors, it is suggested that unaged seeds utilize both respiration pathways in the cotyledon but only the cytochrome pathway in the axis. Accelerated aging causes a marked deterioration of respiration, especially that through the cytochrome pathway, and there is an associated engagement of the alternative pathway in the seed axis. It is suggested that the lowering of respiratory activity and the shift in respiratory pathways may play a major role in the decline of germinability and vigor.     

Spectral evidence for a component involved in hydrogen metabolism of soybean nodule bacteroids

A component with a difference spectrum similar to that of b-type cytochromes which becomes reduced upon the addition of H₂ has been demonstrated in soybean nodule bacteroids. This electron carrier, referred to as component 559-H₂, is present in hydrogenase-positive strains of Rhizobium japonicum but has not been detected in mutants that lack hydrogenase activity or in hydrogenase-negative wild-type strains. A positive correlation between concentrations of component 559-H₂ and hydrogenase activities has been established. These results provide further evidence that component 559-H₂ is involved in H₂ metabolism in R. japonicum.     

Indole-3-acetic acid production by bacteroids from soybean root nodules

Purine nucleotide and RNA synthesis have been investigated at the different growth stages of carrot ( Daucus carota L.) cells grown in suspension cultures. At the early growth stages an increase in the content of RNA was observed, although at later stages RNA was degraded. The highest rates of incorporation of [¹⁴C]-labelled adenosine into ATP and GTP were observed at the late growth sttages. This indicated that purine slavage was more importnt at the late growth stages, while de novo synthesis was dominant during the initial growth stages. This pattern was also reflected by increased levels, in the cell dividison phase, of theenzymes glycinamide ribonucleotide synthetase (EC 6.3.1.3.) and phosphoribosylpyrophosphate amido-transferase (EC 2.4.2.14) involved in de novo purine synthesis. The activities of the purine salvage enzymes varied little during growth. Cells in the stationary phase, that were starved for sucrose and phosphate, showed a dramatic increase in cellular metabolism, as judged from a rapid uptake and incorporation of [³²P]-labelled phosphate into nucleotides and RNA, when incubated in fresh medium.