Changes in the activity and isozyme patterns of malate dehydrogenase in root nodules of yellow lupine

The malate dehydrogenase present in the cytoplasmic fraction of plant origin and bacteroids from yellow lupine root nodules was investigated. The plant enzyme was 14 times more active in nodules than in roots and it contained 6 molecular forms in nodules compared with 3 forms detected in roots. The highest malate dehydrogenase activity in plant fraction and bacteroids was noted in 50-day old plants. Changes in the isoenzymatic patterns of malate dehydrogenase in plant fraction and bacteroids accompanying ageing of the lupine root nodules were observed. Possible physiological role of malate pathway in metabolism of lupine root nodules is discussed.     

Methemoglobin Reductase of Bacteria and Bacteroids <Emphasis Type="Italic">Bradyrhizobium lupini</Emphasis>: Purification and Properties

Reductase capable of reducing hemoglobin-like proteins was isolated from nodule bacteria Bradyrhizobium lupini and bacteroids of lupine root nodules. It is similar in some properties to many known methemoglobin reductases reducing animal and plant hemoglobins. It is a NADH-dependent FAD-containing flavoprotein with molecular weight of 87 kDa without metals. The presence of such enzymes in prokaryotes could be an explanation for the physiological activity of both bacterial and eukaryotic hemoglobins expressed in bacterial cells.     

Nitrate Effect on Nitrogen Fixation (Acetylene Reduction): ACTIVITIES OF LEGUME ROOT NODULES INDUCED BY RHIZOBIA WITH VARIED NITRATE REDUCTASE ACTIVITIES

The effect of nitrate on symbiotic nitrogen fixation by root nodules of cowpea (Vigna unguiculata L., Walp., cv. California Blackeye) and lupine (Lupinus augustifolius L., cv. Frost) plants inoculated with nitrate reductase-expressing and nitrate reductase-nonexpressing Rhizobium strains were examined. Nitrate reductase of Rhizobium bacteroids in the nodules of cowpea and lupine reduced nitrate to nitrite. Both cowpea and lupine nodules accumulated nitrite when grown in the presence of 15 millimolar nitrate and induced by Rhizobium strains which express nitrate reductase activity (Rhizobium sp. 32H1 and 127E15). The nitrogen fixation (acetylene reduction) activities of cowpea and lupine nodules were inhibited by nitrate whether the nodules were induced by Rhizobium strains that express (Rhizobium sp. 32H1 and 127E15) or do not express (Rhizobium sp. 127E14 and R. lupini ATCC 10318) nitrate reductase activity. These findings indicate that nitrite, the product of bacteroid nitrate reductase, may not play a role in the inhibitory effect of nitrate on nitrogen fixation activities of legume root nodules. However, the degree of inhibition on the fixation activity by nitrate varied in different legume-Rhizobium combinations.     

Effect of water stress on the reduction of nitrate and nitrite by soybean nodules

The effects of water stress on nitrate reductase and nitrite reductase activities in symbiotic nodules were examined in field-grown soybean plants (Glycine max L Merr. cv Clark). The in vitro assays of enzyme activity indicated that the nodule cytosol and bacteroids contained both nitrate reductase and nitrite reductase activities. The reduction of nitrate in bacteroids increased significantly as nodule water potential declined from −0.6 to −1.4 megapascals, and then decreased when −1.8 megapascals water potential was reached. On the contrary, the reduction of nitrate in nodule cytosol was inhibited as water stress progressed. Increases in water stress intensity also caused a significant inhibition in nitrite reductase activities of bacteroids and nodule cytosol within soybean nodules. The results show that nitrate reduction occurred both in the cytosol and bacteroids of water-stressed soybean nodules. The reduction of nitrate functioned at different physiological modes in these two fractions.     

Alcohol dehydrogenase and its relation to respiratory pathways in lupine root nodules.

Changes in the isoenzymatic patterns of alcohol dehydrogenase (EC 1.1.1.1) accompanying ageing of the lupine root nodules were observed. Ethanol and other products of anaerobic metabolic pathways (lactate and malate) are better respiratory substrates for bacteroids and symbiosomes (peribacteroid units, PBUs) than glucose and pyruvate. It is postulated that fermentative processes in lupine root nodule provide energy and substrates for bacteroids.     

Growth of nodule bacteria in soil]

As was found with the aid of capillary microscopy, the nodule bacteria of pea and lupine in soil are encountered as rods, bacteroids, and cocci. The rod-like cells form bacteroids not only in the nodules but also outside, in soil. The bacteroids are viable and later reproduce the coccoid cells (arthrospores). The rods and bacteroids prevail in soil during flowering of the legumes, and the cocci are predominant at the end of vegetation.     

Main centers of nitrate and nitrite reduction in young and nodulated yellow lupine (<Emphasis Type="Italic">Lupinus luteus</Emphasis>)

Nitrate and nitrite reduction centers in non-nodulated and symbiotic yellow lupine were analyzed. In young seedlings, nitrate was exclusively accumulated in roots, which also was shown as the main nitrate reduction center. In contrast, leaves were shown to play a key role in nitrite reduction. A similar distribution of nitrate reductase (NR) and nitrite reductase was found in nodulated plants. However, in field conditions characterized by low nitrate content, a disproportionately high level of NR activity in nodules was also observed during all stages of symbiotic growth. This feature was confirmed in nitrate-fed hydroponic cultures. Nodule NR activity was one order of magnitude higher than in roots, in spite of the small stored nitrate pool found inside nodules. This suggests that nodule NR activity had been induced not by nitrate itself but indirectly. Since bacteroids were shown to be responsible for the vast majority of nodule NR activity, the plausible explanation of this effect seems to be a dissimilatory nature of rhizobial NR. Considering that environmental nitrate could cause hypoxia inside nodules, this is the proposed way of the observed nodule NR induction.     

Labelling of lupine nodule metabolites with 14CO2 assimilated from the leaves

On feeding ¹⁴CO₂ to the shoots of lupine (25 mCi per plant) 30 min was the minimal time needed to determine the incorporation of label into bacteroid compounds. The predominant incorporation, exhibited in all root, nodule and bacteroid samples after 30 min exposure, was into sucrose (45–90% of the corresponding fraction radioactivity) of the neutral fraction; into malate (30–40%) of the acid fraction; into aspartic acid and asparagine (60–80% in sum) of the basic fraction. The composition of carbon compounds containing the greatest amount of ¹⁴C in the cytosol of nodules and in bacteroids was similar. Their radioactivity after 30 min exposure was for bacteroids (nCi per g of bacteroid fr. wt): sucrose 5.73, glucose 1.00, malate 0.15, succinate 0.11; for the nodule cytosol (nCi per g of nodule fr. wt): sucrose 200.00, glucose 8.40, malate 9.34, succinate 8.50. Thus it was demonstrated that in lupine, sucrose is the main photoassimilate entering not only into nodules but also into bacteroids. The biosynthesis of aspartic acid and asparagine occurs during nitrogen fixation in bacteroids.     

Nitrogen fixation in transposon mutants from Bradyrhizobium japonicum USDA 110 impaired in nitrate reductase

Tn5 transposon mutagenesis was carried out in Bradyrhizobium japonicum strain USDA 110 to produce defective mutants. From over one thousand clones expressing low levels of nitrate reductase activity as free-living bacteria, approximately five percent had significantly different ratios of nodulation, N2 fixation or nitrate reductase activity compared to the wild strain when determined in bacteroids from soybean nodules. Tn5 insertions were checked previously and mutants were arranged into four different groups. Only one of these groups, designated AN, was less effective at N2 fixation than the wild strain, suggesting a mutation in a domain shared by nitrogenase and NR. The remaining groups of insertions successfully nodulated and were as effective at N2 fixation as the wild strain, but showed diminished ability to reduce nitrate both in nodules and in the isolated bacteroids when assayed in vitro with NADH or methyl viologen as electron donors. PCR amplification demonstrated that Tn5 insertions took place in different genes on each mutant group and the type of mutant (CC) expressing almost no nitrate reductase activity under all treatments seemed to possess transposable elements in two genes. Induction of nitrate reductase activity by nitrate was observed only in those clones expressing a low constitutive activity (AN and AE). Nitrate reductase activity in bacteroids along nodule growth decreased in all groups including the ineffective AN group, whose nodulation was highly inhibited by nitrate at 5 mmol/L N. Host-cultivar interaction seemed to influence the regulation of nitrate reductase activity in bacteroids. Total or partial repression of nitrate reductase activity in bacteroids unaffected by N2 fixation (CC, AJ and AE groups) improved nodule resistance to nitrate and N yields of shoots over those of the wild strain. These observations may suggest that some of the energy supplied to bacteroids was wasted by its constitutive NRA.     

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.     

14C-glucose utilization byRhizobium lupini bacteroids

Summary The ability of bacteroids, isolated fromLupinus luteus L. nodules at the stage of active nitrogen fixation, to assimilate (1-¹⁴C)-glucose and (2-¹⁴C)-glucose was being studied. The label is incorporated into all the Krebs cycle metabolites, amino acids and sugars after 5 min of glucose insertion into cell suspension. High activity of glucose phosphorylation was found in bacteroidsin vitro, the reaction rate being the highest at a glucose concentration of over 100 mM.In lupine nodules sugars can be essential carbon substrate delivered to the bacteroids from host-plant cells. This point of view is discussed.     

Nitrate reductase from bacteroides of Rhizobium japonicum: enzyme characteristics and possible interaction with nitrogen fixation.

The soluble nitrate reductase of Rhizobium japonicum bacteroids has been purified and its properties compared to those of aerobically grown cells. The enzymes from both sources are similar with molecular weights of about 70 000 suggesting no close relationship with the molybdo-protein component of nitrogenase. Nitrite, the product of nitrate reductase, strongly inhibited the nitrogenase activity from bacteroids, at concentrations less than 100 muM. Thus, an interference in the rate of nitrogen fixation is possible as a result of nitrate reductase activity. A study of the distribution of nitrate reductase in bacteroids indicates that a proportion of the total activity is membrane-bound but that this activity is similar to that in the soluble fraction. Purified nitrate reductase required reduced viologen dyes for activity. Neither NADPH or NADH or FAD could substitute as electron donors. Dithionite is a strong inhibitor and inactivated nitrate reductase from all sources examined. This inactivation is prevented by methyl viologen. Purified nitrate reductase from bacteroids and bacteria Rhizobium japonicum is practically unaffected by exposure to oxygen.     

Nitrate reductase activities of rhizobia and the correlation between nitrate reduction and nitrogen fixation.

All species of Rhizobium except R. lupini had nitrate reductase activity. Only R. lupini was incapable of growth with nitrate as the sole source of nitrogen. However, the conditions necessary for the induction of nitrate reductase varied among species of Rhizobium. Rhizobium japonicum and some Rhizobium species of the cowpea strains expressed nitrate reductase activities both in the root nodules of appropriate leguminous hosts and when grown in the presence of nitrate. Rhizobium trifolii, R. phaseoli, and R. leguminosarum did not express nitrate reductase activities in the root nodules, but they did express them when grown in the presence of nitrate. In bacteroids of R. japonicum and some strains of cowpea Rhizobium, high N2 fixation activities were accompanied by high nitrate reductase activities. In bacteroids of R. trifolii, R. leguminosarum, and R. phaseoli, high N2 fixation activities were not accompanied by high nitrate reductase activities.     

Nitrate reductase and nitrite reductase activity in free-living cells and bacteroids of Rhizobium loti

Cells of Rhizobium loti strains T1 and U226 cultured in defined medium with glutamate as the only nitrogen source and bacteroids isolated from root nodules of Lotus corniculatus, L. pedunculatus and L. tenuis did not show constitutive (non-nitrate induced) nitrate reductase activity (NRA). In contrast, nitrite reductase activity (NiRA) was present in both free-living cells and bacteroids of either strain T1 or U226. Constitutive NRA and NiRA were detected in the cytosol fraction from nodules of all three symbioses examined. An induced NRA was expressed in bacteroids after a 10 h incubation in the presence of nitrate.     

Adenosine-3':5'-monophosphate phosphodiesterase from Rhizobium

The phosphodiesterase (PDE) activity of adenosine-3':5'-monophosphate was detected in the cells of tubercular bacteria of Rhizobium lupini and Rhizobium japonicum. The specific activity of three Rhizobium forms, e.g. bacteroids from lupine root tubercles, free-nitrogen-fixing culture and vegetative cells grown on a mannitol--yeast agar, were compared. In the bacteroids PDE is represented both by soluble and membrane-bound forms. The optimal enzyme activity is revealed in an alkaline medium, whereas the curve of PDE activity dependence on pH has a broad maximum. PDE is inhibited by methylxanthines, the inhibiting effect being stronger than that of theophylline.     

The arginine deiminase pathway in Rhizobium etli: DNA sequence analysis and functional study of the arcABC genes.

Sequence analysis upstream of the Rhizobium etli fixLJ homologous genes revealed the presence of three open reading frames homologous to the arcABC genes of Pseudomonas aeruginosa. The P. aeruginosa arcABC genes code for the enzymes of the arginine deiminase pathway: arginine deiminase, catabolic ornithine carbamoyltransferase (cOTCase), and carbamate kinase. OTCase activities were measured in free-living R. etli cells and in bacteroids isolated from bean nodules. OTCase activity in free-living cells was observed at a different pH optimum than OTCase activity in bacteroids, suggesting the presence of two enzymes with different characteristics and different expression patterns of the corresponding genes. The characteristics of the OTCase isolated from the bacteroids were studied in further detail and were shown to be similar to the properties of the cOTCase of P. aeruginosa. The enzyme has a pH optimum of 6.8 and a molecular mass of approximately 450 kDa, is characterized by a sigmoidal carbamoyl phosphate saturation curve, and exhibits a cooperativity for carbamoyl phosphate. R. etli arcA mutants, with polar effects on arcB and arcC, were constructed by insertion mutagenesis. Bean nodules induced by arcA mutants were still able to fix nitrogen but showed a significantly lower acetylene reduction activity than nodules induced by the wild type. No significant differences in nodule dry weight, plant dry weight, and number of nodules were found between the wild type and the mutants. Determination of the OTCase activity in extracts from bacteroids revealed a strong decrease in activity of this enzyme in the arcA mutant compared to the wild-type strain. Finally, we observed that expression of an R. etli arcA-gusA fusion was strongly induced under anaerobic conditions.     

Effect of low nitrate supply to nodulated lucerne on time course of activites of enzymes involved in inorganic nitrogen metabolism

Plants of lucerne ( Medicago sativa L. cv. Aragón) inoculated with several strains of Rhizobium meliloti were supplied with a low level of nitrate (5 m M ). After 1 week, normalised nodule mass, obtained by dividing nodule weight by shoot weight, was decreased by one-fourth. This result closely paralleled the bacteroid protein content of nodules, whereas the cytosolic content remained constant. Nitrate reductase activity (NRA, EC 1.7.99.4) of bacteroids increased rapidly after nitrate supply, with actual rates being highly dependent on the Rhizobium strain. The expression of cytosolic NR (EC 1.6.6.1) also varied depending on the bacterial strain but was largely insensitive to nitrate feeding. Nitrite reductase activity (NiRA, EC 1.7.2.2) of either bacteroid or plant origin was independent of the R. meliloti strain. Activation occurred after 3 and 7 days, respectively, of nitrate feeding. Significant amounts of nitrite were obtained throughout the experimental period from buffered extracts of both bacteroids and cytosol of nodules. However, when these nodules were ground in the presence of inhibitors of enzyme activity, nitrite was only found in nodules containing strain 102-F-51 after 1 week of treatment. These results agree with the recent hypothesis that nitrite plays a role in a secondary stage of nodule damage by nitrate. We propose that NiRA rather than NRA can be used as an internal probe of nitrate access to the infected region of nodules.     

Denitrification in lucerne nodules is not involved in nitrite detoxification

Dissimilatory reduction of ionic nitrogen oxides to gaseous forms such as nitrous oxide or nitrogen can be carried out by free living or symbiotic forms of some strains of Rhizobium meliloti. In this paper we investigate whether bacteroid denitrification plays a role in the alleviation of the inhibitory effects of nitrate on nitrogen fixation both in bacteroid incubations as in whole nodules. The presence of a constitutive nitrate reductase (NR) activity in isolated bacteroids caused nitrite accumulation in the incubation medium, and acetylene reduction activity in these bacteroids was progressively inhibited, since nitrite reductase (NiR) activity was unable to reduce all the nitrite produced by NR and denitrification occurred slowly. Even nodules infiltrated with nitrate and nitrite failed to increase gaseous forms of nitrogen substantially, indicating that nitrite availability was not limiting denitrification by bacteroids. In spite of the low rates of bacteroidal denitrification, the effect of nodule denitrification on the inhibition of nitrogen fixation by nitrate in whole plants was tested. For that purpose, lucerne plants (Medicago sativa L. cv. Aragon) were inoculated with two Rhizobium meliloti strains: 102-F-65 (non denitrifying) and 102-F-51 (a highly denitrifying strain). After a seven days nitrate treatment, both strains showed the same pattern of inhibition, and it occurred before any nitrate or nitrite accumulation within the nodules could be detected. This observation, together with the lack of alleviation of the ARA inhibition in the denitrifying strain, and the limited activity of dissimilatory nitrogen reduction present in these bacteroids, indicate a role other than nitrite detoxification for denitrification in nodules under natural conditions.     

Nitrate and Nitrite Reduction in Relation to Nitrogenase Activity in Soybean Nodules and Rhizobium japonicum Bacteroids

Soybean (Glycine max L. cv Williams) seeds were sown in pots containing a 1:1 perlite-vermiculite mixture and grown under greenhouse conditions. Nodules were initiated with a nitrate reductase expressing strain of Rhizobium japonicum, USDA 110, or with nitrate reductase nonexpressing mutants (NR⁻ 108, NR⁻ 303) derived from USDA 110. Nodules initiated with either type of strain were normal in appearance and demonstrated nitrogenase activity (acetylene reduction). The in vivo nitrate reductase activity of N₂-grown nodules initiated with nitrate reductase-negative mutant strains was less than 10% of the activity shown by nodules initiated with the wild-type strain. Regardless of the bacterial strain used for inoculation, the nodule cytosol and the cell-free extracts of the leaves contained both nitrate reductase and nitrite reductase activities. The wild-type bacteroids contained nitrate reductase but not nitrite reductase activity while the bacteroids of strains NR⁻ 108 and NR⁻ 303 contained neither nitrate reductase nor nitrite reductase activities.

Addition of 20 millimolar KNO₃ to bacteroids of the wild-type strain caused a decrease in nitrogenase activity by more than 50%, but the nitrate reductase-negative strains were insensitive to nitrate. The nitrogenase activity of detached nodules initiated with the nitrate reductase-negative mutant strains was less affected by the KNO₃ treatment as compared to the wild-type strain; however, the results were less conclusive than those obtained with the isolated bacteroids.

The addition of either KNO₃ or KNO₂ to detached nodules (wild type) suspended in a semisolid agar nutrient medium caused an inhibition of nitrogenase activity of 50% and 65% as compared to the minus N controls, and provided direct evidence for a localized effect of nitrate and nitrite at the nodule level. Addition of 0.1 millimolar sucrose stimulated nitrogenase activity in the presence or absence of nitrate or nitrite. The sucrose treatment also helped to decrease the level of nitrite accumulated within the nodules.