Hydrogenase in pea root nodule bacteriods

Summary Hydrogen uptake has been shown to occur with pea root nodule breis and this uptake has been shown to be confined to the bacteriods. The uptake of hydrogen by washed bacteriods, in the absence of any added substrates, has been shown to be accompanied by oxygen uptake and the ratio of hydrogen uptake to oxygen uptake in these preparations has been found to be 2:1. Substrates, provided to washed bacteriods, inhibit the uptake of hydrogen and it has been found that the utilisation of substrates, as measured by carbon dioxide evolution, is inhibited by hydrogen. It is suggested that hydrogen and substrates compete for electron carriers and that electrons from the hydrogen reduce components of the electron transport pathway and ATP is produced.The action of hydrogen on nitrogen fixation in nodule breis and washed bacterioid preparations was examined and the evidence shows that some non-competitive inhibition of nitrogen fixation, by hydrogen, occurs.     

Hydrogenase in actinorhizal root nodules and root nodule homogenates.

Hydrogenases were measured in intact actinorhizal root nodules and from disrupted nodules of Alnus glutinosa, Alnus rhombifolia, Alnus rubra, and Myrica pensylvanica. Whole nodules took up H2 in an O2-dependent reaction. Endophyte preparations oxidized H2 through the oxyhydrogen reaction, but rates were enhanced when hydrogen uptake was coupled to artificial electron acceptors. Oxygen inhibited artifical acceptor-dependent H2 uptake. The hydrogenase system from M. pensylvanica had a different pattern of coupling to various electron acceptors than the hydrogenase systems from the alders; only the bayberry system evolved H2 from reduced viologen dyes.     

Surprising developments in the legume root nodule

Root nodule morphogenesis involves the induction of mitotic activity in otherwise quiescent root cortical cells, giving rise to the nodule primordium. One gene expressed during nodule initiation, ENOD40, has been implicated in nodule growth and/or differentiation^(1,2). Interestingly, although the nucleotide sequence of ENOD40 genes from soybean^(3,4) and Medicago^(1,2) are highly homologous, they are unlikely to encode a similar protein product. In fact, a remarkable feature of these genes is their apparent lack of protein coding potential. Thus, ENOD40 is a member of the growing list of eukaryotic genes whose RNA product is implicated in control of cell growth and differentiation, the so called riboregulators⁽⁵⁾.     

Occurrence and some properties of carbonic anhydrases from legume root nodules

Carbonic anhydrase activity (hydration of CO₂ was found in homogenates of leaves (116–500 units.mg^?1 protein) and root nodules (27–255 units.mg^?1 protein) from 8 legume genera inoculated in each case with a host specific Rhizobium. No enzyme, or only trace amounts (2–7 units.mg^?1 protein), were detected in root extracts, The enzymatic activity was inhibited in all cases by azide and acetazolamide. The sizes of nodule and leaf carbonic anhydrases, estimated by gel filtration of partially purified preparations from Phaseolus vulgaris, were around 45 000 and 205 000 respectively. These enzymes also differed in sensitivity to inhibitors. More than 99% of the activity present in Vicia faba nodules was recovered as a soluble enzyme and only a trace was located in the isolated bacteroids.     

DNA synthesis and fragmentation in bacteroids during Astragalus sinicus root nodule development

Histo- and cytochemical techniques were used to study the DNA replication and fragmentation patterns in bacteroids formed by Mesorhizobium huakuii subsp. rengei in nodules of Astragalus sinicus. DNA replication was detected by the incorporation of 5-bromo deoxy-uridine. Signals denoting DNA synthesis were observed in plant nuclei within the nodule meristem and in bacteroids near the meristem. The TUNEL (TdT-mediated dUTP nick-end labeling) assay was used to measure DNA fragmentation. In nutrient-depleted 1-mpi (month(s) post inoculation) nodule sections, some bacteroids were in vacuoles, and DNA fragmentation signals were observed only in such bacteroids. In contrast, 1-mpi nodule sections without nutrient depletion showed neither bacteroid localization in vacuoles nor DNA fragmentation signals. The bacteroid translocation into vacuoles upon nutrient starvation might results from autophagy of the plant. In 2-mpi nodule sections, bacteroids with DNA fragmentation signals appeared within the cytoplasm of some nodule cells in the senescence zone.     

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     

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.     

Lipopolysaccharide epitope expression of Rhizobium bacteroids as revealed by in situ immunolabelling of pea root nodule sections.

To investigate the in situ expression of lipopolysaccharide (LPS) epitopes on nodule bacteria of Rhizobium leguminosarum, monoclonal antibodies recognizing LPS macromolecules were used for immunocytochemical staining of pea nodule tissue. Many LPS epitopes were constitutively expressed, and the corresponding antibodies reacted in nodule sections with bacteria at all stages of tissue infection and cell invasion. Some antibodies, however, recognized epitopes that were only expressed in particular regions of the nodule. Two general patterns of regulated LPS epitope expression could be distinguished on longitudinal sections of nodules. A radial pattern probably reflected the local physiological conditions experienced by endosymbiotic bacteria as a result of oxygen diffusion into the nodule tissue. The other pattern of expression, which followed a linear axis of symmetry along a longitudinal section of the pea nodule, was apparently associated with the differentiation of nodule bacteria and the development of the nitrogen-fixing capacity in bacteroids. Basically similar patterns of LPS epitope expression were observed for pea nodules harboring either of two immunologically distinct strains of R. leguminosarum bv. viciae, although these epitopes were recognized by different sets of strain-specific monoclonal antibodies. Furthermore, LPS epitope expression of rhizobia in pea nodules was compared with that of equivalent strains in nodules of French bean (Phaseolus vulgaris). From these observations, it is suggested that structural modifications of Rhizobium LPS may play an important role in the adaptation of endosymbiotic rhizobia to the surrounding microenvironment.     

Mutual host plant relationships in two groups of legume root nodule bacteria (Rhizobium spp.)

Summary Eighteen strains of Rhizobium lupini were shown to form effectively nitrogen-fixing root nodules in Lotus uliginosus whose rhizobia appeared culturally similar to R. lupini. Six strains formed effective or (in one case) semi-effective nodules in Ornithopus sativus.Evidence has been found for the existence of an extensive cross-inoculation group involving the genera Lupinus, Ornithopus, Lotus, Anthyllis, Caragana, Astragalus, Ononis, Genista, Mimosa and probably others. The rhizobia include fastgrowing as well as slow-growing strains.Dedicated to Professor C. B. Van Niel on the occasion of his 70 th birthday.     

Characterization of three soluble c-type cytochromes isolated from soybean root nodule bacteroids of Bradyrhizobium japonicum strain CC705

Three soluble, low molecular mass cytochromes c (Mr 8000-15,000) were isolated and purified from soybean root nodule bacteroids of Bradyrhizobium japonicum strain CC705. On the basis of their alpha: absorbance peaks in the reduced forms, they were named cytochromes c550, c552 and c555. Cytochrome c552 reacted very fast, c555 very slowly and c550 not at all with carbon monoxide. The complete amino acid sequence (73 residues) of cytochrome c552 was established which identifies it as a monoheme, class I cytochrome c with some remote similarity to the cytochrome c6 family.     

Glutamate synthase in Medicago sativa L. Occurrence and properties of FD-dependent enzyme in plant cell fraction during root nodule development

In the plant cell fraction of Medicago sativa (L. cv Europe) nodules, glutamate synthase is active with reduced Fd, MV, NADH and NADPH as an electron donor. Up to 25 to 30 days after inoculation, the activities of Fd-dependent glutamate synthase (EC 1.4.1.7), the most active form of the enzyme, NADH-dependent (EC 1.4.1.14) and NADPH-dependent (EC 1.4.1.13) glutamate synthases increase about 2-fold followed by a relatively constant level per gram fresh weight of nodules. The activities of glutamate synthases with different electron carriers increase constantly about 30-fold after 46 days of inoculation by total fresh weight of nodules per plant. These nodule glutamate synthase activities with Fd, NADH or NADPH represent 30% relative to those of root glutamate synthases per plant with the respective electron donor. Fd-glutamate synthase in nodule plant fraction is a protein molecule immunochemically distinct from pyridine nucleotide-glutamate synthases. MV-linked enzyme activity is associated with Fd-glutamate synthase. The Fd-glutamate synthase has a subunit molecular mass of 68.2 kDa, and it exhibits a high affinity for spinach Fd as an electron carrier. The increase in Fd-glutamate synthase activity during nodule development is accompanied by a rise in the enzyme protein content. The total activity of different forms of glutamate synthase in vitro ensures a higher level than the rate of ammonia production during N2 fixation in bacteroids of Medicago sativa nodules.     

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     

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.