Developmental fate of Rhizobium meliloti bacteroids in alfalfa nodules.

Nitrogen-fixing bacteroids are degraded during nodule senescence. This is in contrast to recent implications that viable bacteroids can be released into soil from legume nodules. Rhizobia originating from persistent infection threads in senescing nodule plant cells seem to be the source of viable cells required for perpetuation of the Rhizobium spp. population in the soil. Our conclusions were derived from electron microscopic examination of stages of development and senescence of alfalfa root nodules.     

Nitrate and nitrite reduction by alfalfa root nodules: Accumulation of nitrite in Rhizobium melioti bacteroids and senescence of nodules

Addition of NO⁻₃ rapidly induced senescence of root nodules in alfalfa ( Medicago sativa L. cv. Aragon). Loss of nodule dry matter began at the lowest NO⁻₃ concentration (10 m M ) but degradation of bacteroid proteins was only detected when nodules were supplied with NO⁻₃ concentrations above 20 m M .
Bacteroids from Rhizobium meliloti contained high specific activities of nitrate reductase (NR) and nitrite reductase (NiR). Both enzymes were presumably substrate-induced although substantial enzyme activities were present in the absence of NO⁻₃ Typical specific activities for soluble NR and NiR of bacteroids under NO⁻₃ free conditions were 1.2 and 1.4 μmol (mg protein)⁻¹h⁻¹, respectively. In the presence of NO⁻₃, the specific activity of NR was considerably greater than that of NiR, thus causing NO⁻₂ accumulation in bacteroids. Nitrite levels in the bacteroids were linearly correlated with specific activities of NR and NiR, indicating that NO⁻₂ is formed by bacteroid NR and that this NO⁻₂ in turn, induces bacteroid NiR. Accumulation of NO⁻₂ within bacteroids also indicates that NO⁻₂ inhibits nodule activity after feeding plants with NO⁻₃     

Involvement of the cytoplasmic membrane in nitrogen fixation by Rhizobium leguminosarum bacteroids

1. The nitrogen-fixing efficiency of freshly prepared suspensions of Rhizobium leguminosarum bacteroids from pea root nodules was considerably enhanced by addition of bovine serum albumin. Evidence was found that during preparation of bacteroids the cell membrane is exposed to the uncoupling effect of free fatty acids and to plant phospholipase D activity. Both effects could be counteracted by bovine serum albumin. 2. A technique was developed by which concentrations of free O2 and nitrogenase activity could be measured simultaneously under conditions of steady-state respiration. By means of this system it could be shown that in contrast to previous claims, high ATP/ADP ratios can be achieved in bacteroids even with a high concentration of O2 in the medium. 3. Nitrogen fixation was found to be controlled by the ATP/ADP ratio, the generation of reducing equivalents and the switch-off phenomenon. It was demonstrated that the generation of reducing equivalents for nitrogenase is regulated by the energized state and the integrity of the bacteroid cell membrane. The data indicate that the process of aerobic nitrogen fixation in R. leguminosarum bacteroids resembles that of Azotobacter vinelandii.     

Regulation of symbiotic nitrogen fixation in root nodules of alfalfa (Medicago sativa) infected with Rhizobium meliloti

Symbiotic nitrogen fixation of Rhizobium meliloti bacteroids in Medicago sativa root nodules was suppressed by several inorganic nitrogen sources. Amino acids like glutamine, glutamic acid and aspartic acid, which can serve as sole nitrogen sources for the unnodulated plant did not influence nitrogenase activity of effective nodules, even at high concentrations.Ammonia and nitrate suppressed symbiotic nitrogen fixation in vivo only at concentrations much higher than those needed for suppression of nitrogenase activity in free living nitrogen fixing bacteria. The kinetics of suppression were slow compared with that of free living nitrogen fixing bacteria. On the other hand, nitrite, which acts as a direct inhibitor of nitrogenase, suppressed very quickly and at low concentrations. Glutamic acid and glutamine enhanced the effect of ammonia dramatically, while the suppression by nitrate was enhanced only slightly.     

The Lotus japonicus Sen1 gene controls rhizobial differentiation into nitrogen-fixing bacteroids in nodules

A Lotus japonicus mutant, Ljsym75, which forms ineffective symbiotic nodules and defines a new locus involved in the process of nitrogen fixation, was characterized in detail in order to identify the stage of developmental arrest of the nodules. No nitrogen-fixing activity was detectable in Ljsym75 nodules at any stage during plant development, and plant growth was markedly retarded. Ljsym75 plants formed twice as many nodules as the wild-type Gifu, and this phenotype was not influenced by the application of low concentrations of nitrate. Although the ineffective nodules formed on Ljsym75 were anatomically similar to effective Gifu nodules, Ljsym75 nodules senesced prematurely. Microscopic examination revealed that bacteria endocytosed into Ljsym75 nodules failed to differentiate into bacteroids. Moreover, the bacteria contained no nitrogenase proteins, whereas leghemoglobin was detected in the cytosol of the nodules. These results indicate that Ljsym75 is required for bacterial differentiation into nitrogen-fixing bacteroids in nodules, and thus the Ljsym75 gene was renamed sen1 (for stationary endosymbiont nodule). Linkage analysis using DNA markers showed that Sen1 is located on chromosome 4.     

Relationships between nitrogen fixation and photosynthesis as the main components of the productivity in alfalfa

Alfalfa (Medicago sativa L.) plants were inoculated with Sinorhizobium meliloti Tn-5 mutants featuring various nitrogen-fixing effectiveness and then grown in sand culture to study relations between CO₂ exchange, plant productivity, and nitrogen fixation. At the flowering stage, the relationship between nitrogen fixation and photosynthesis of whole alfalfa plants was described with the logarithmic curve. At the same stage of plant development, a close relationship was observed between nitrogen fixation rate and plant weight; this relationship showed a trend toward saturation at high rates of nitrogen fixation. The increase in nitrogenase activity of root nodules was accompanied by stimulation of root respiration; the relation of respiration to nitrogen-fixing activity was manifested stronger than its relation to the total root weight. It is concluded that highly effective strains of root nodule bacteria can realize their potential only in combination with complementary plant genotypes featuring active photosynthesis that provides a balanced supply of assimilates for both the symbiotic apparatus and growth processes in the macrosymbiont.     

Nitrogen fixation and carbon metabolism by nodules and bacteroids of pea plants under sodium chloride stress

Acetylene reduction activity (ARA) and leghemoglobin (Lb) content in nodules were sigificantly reduced when pea ( Pisum sativum L. cv. Lincoln) plants were subjected to 50 m M sodium chloride stress for 3 weeks. C₂H₂ reduction activity by bacteriods isolated from pea nodules was drastically inhibited by saline stress, and malate appeared to be a more appropriate substrate than glucose or succinate in maintaining this activity. Salt added directly to the incubation mixture of bacteriods or to the culture medium of plants inhibited O₂ uptake by bacteroids. Nodule cytosolic phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) and bacteriod malate dehydrogenase (MDH; EC 1.1.1.37) activities were strongly enhanced by salt stress. Under these conditions, malate concentration was depressed in bacteroids and cytosol, whereas total soluble sugar (TSS)content slightly increased in both fractions. The effect of salt stress on TSS and malate content suggests that the utilization of carbohydrate within nodules could be inhibited during salt stress. The inhibitory effect of NaCl on N₂ fixation activity of bacteroids and to the decrease in bacteroid respiration. The stimulation of fermentative metabolism induced by salinity suggests some reduction in O₂ availability within the nodule. Salt stress was also responsible for a decrease of the cytosolic protein content, specifically of leghemoglobin, in the nodules.     

Oxygen and the regulation of nitrogen fixation in legume nodules

In N₂-fixing legume nodules, O₂ is required in large amounts for aerobic respiration, yet nitrogenase, the bacterial enzyme that fixes N₂, is O₂ labile. A high rate of O₂ consumptition and a cortical barrier to gas diffusion work together to maintain a low, non-inhibitory O₂ concentration in the central, infected zone of the nodule. At this low O₂ concentration, cytosolic leghemoglobin is required to facilitate the diffusion of O₂ through the infected cell to the bacteria. The resistance of the cortical diffusion barrier is variable and is used by legume nodules to regulate the O₂ concentration in the infected cells such that it limits aerobic respiration and N₂ fixation at all times. The resistance of the diffusion barrier and therefore the degree of O₂ limitation seems to be regulated in response to changes in the O₂ concentration of the central infected zone, the supply of phloem sap to the nodule, and the rate of N assimilation into the end products of fixation.     

The Rhizobium etli gene iscN is highly expressed in bacteroids and required for nitrogen fixation

Sequence analysis of the rpoN (2)- fixA intergenic region in the genome of Rhizobium etli CNPAF512 has uncovered three genes involved in nitrogen fixation, namely nifU, nifS and nifW. These genes are preceded by an ORF that is highly conserved among nitrogen-fixing bacteria. It encodes a putative gene product of 105 amino acids, belonging to the HesB-like protein family. A phylogenetic analysis of members of the HesB-like protein family showed that the R. etli HesB-like protein clusters with polypeptides encoded by ORFs situated upstream of the nifUS nitrogen fixation regions in the genomes of other diazotrophs. The R. etli ORF that encodes the HesB-like protein was designated iscN. iscN is co-transcribed with nifU and nifS, and is preferentially expressed under free-living microaerobic conditions and in bacteroids. Expression is regulated by the alternative sigma factor RpoN and the enchancer-binding protein NifA. A R. etli iscN mutant displays a reduction in nitrogen fixation capacity of 90% compared to the wild-type strain. This Nif(-) phenotype could be complemented by the introduction of intact copies of R. etli iscN.     

Water relations and nitrogen fixation in potassium fedVigna radiata nodules

Drought created by withholding the irrigation at 30 and 45 d after sowing significantly decreased relative water content (RWC) and osmotic potential (ψ_s) ofVigna radiata (L.) Wilczek cv. MH-83-30 nodules. Potassium fed plants showed higher RWC, whereas ψ_s was further declined irrespective of soil moisture levels. The nitrogenase activity and leghemoglobin content of nodules markedly decreased under drought and nodules of potassium fed plants showed better recovery after rehydration. The proline content significantly increased under drought but declined upon reirrigation. Also, the C, N and K contents of nodules significantly declined under drought.     

Denitrification in lucerne nodules and bacteroids supplied with nitrate

Nodulated lucerne plants ( Medicago sativa L. cv. Aragón) were supplied with 20 m M nitrate. Anaerobically isolated bacteroids of Rhizobium meliloti from these plants were able to denitrify after 48 h treatment. R. meliloti bacteroids behave as total denitrifiers, reducing nitrate to dinitrogen: when acetylene was omitted from the assay medium very little nitrous oxide was recovered. The onset of denitrification activity was coincident with the induction of nitrite reductase activity (EC 1.7.99.3) whereas nitrate reductase activity (EC 1.7.99.4) was constitutive. Whole nodules from plants receiving several doses of nitrate were assayed, in a nitrate-free medium, to monitor denitrification activity dependent on nitrate within the nodules. Denitrification activity was detected after 2 days of 20 m M nitrate supply or after 3 days in the presence of 10 or 5 m M nitrate. These results are discussed in relation to current controversy about nitrate entry into the infection region of nodules. It is concluded that this process occurs more rapidly than suggested in recent research.     

The relationship between nitrogen fixation and the production of HD from D2 by cell-free extracts of soya-bean nodule bacteroids

1. Cell-free extracts prepared from soya-bean nodule bacteroids produced HD from D₂ in the presence of dithionite, an ATP-generating system and nitrogen. 2. Crude extracts of bacteroids or of Azotobacter vinelandii showed some background D₂ exchange when any one of these was omitted. 3. Partial purification of bacteroid extracts diminished this background activity and gave increased D₂ exchange and nitrogen fixation. 4. Although increasing pN₂ stimulated both reactions, the apparent K_m (N₂) for nitrogen fixation was much higher than the apparent K_m (N₂) for D₂ exchange when partially purified bacteroid extracts were used. 5. Carbon monoxide was a competitive inhibitor of nitrogen fixation by partially purified bacteroid extracts, but D₂ exchange was inhibited in a non-competitive fashion. 6. These results are discussed in relation to the possible existence of enzyme-bound intermediates of nitrogen fixation.     

The relative efficiency of nitrogen fixation in pea root nodules

Summary Experiments were performed to investigate the causes of low relative efficiency, RE, in legume root nodules. Nitrogen fixing activity and RE varied with time of incubation of nodules and with different temperatures and oxygen concentrations. The effects of nitrogen concentration and carbon dioxide concentration were also examined. In each case the RE was inversely related to nitrogen fixing activity; measured by acetylene reduction. Increasing the nitrogen concentration had no effect on either nitrogen fixing activity or RE. Experiments with isolated bacteroids gave higher RE values than the whole nodules from which they were isolated. All the results were consistent with hydrogen inhibition of nitrogenase within the nodule being the cause of low RE.     

Effects of drought on nitrogen fixation in soybean root nodules

Soybean plants [Glycine max (L.) Merr.] were grown in silica sand and were drought stressed for a 4 week period during reproductive development and without any mineral N supply in order to maximize demand for fixed nitrogen. A strain of Bradyrhizobium japonicum that forms large quantities of polysaccharide in nodules was used to determine whether or not the supply of reduced carbon to bacteroids limits nitrogenase activity. A depression of 30–40% in nitrogen content in leaves and pods of stressed plants indicated a marked decline in nitrogen fixation activity during the drought period. A 50% increase in the accumulation of bacterial polysaccharide in nodules accompanied this major decrease in nitrogen fixation activity and this result indicates that the negative impact of drought on nodules was not due to a depression of carbon supply to bacteroids. The drought treatment resulted in a statistically significant increase in N concentration in leaves and pods. Because N concentration and chlorophyll concentration in leaves were not depressed, there was no evidence of nitrogen deficiency in drought‐stressed plants, and this result indicates that the negative impact of drought on nodule function was not the cause of the depression of shoot growth. At the end of the drought period, the concentration of carbohydrates, amino nitrogen, and ureides was significantly increased in nodules on drought‐stressed plants. The overall results support the view that, under drought conditions, nitrogen fixation activity in nodules was depressed because demand for fixed N to support growth was lower.     

Metabolism of poly-\-hydroxybutyric acid in bacteroids ofRhizobium lupini in connection with nitrogen fixation and photosynthesis

Summary The darkening of lupin plants grown in a sand culture on a nitrogen-free medium at a stage of initial flowering led to a sharply decreased nitrogen fixation intensity which eventually ceased. Decreased intensity of nitrogen fixation in bacteroids was accompanied by an accumulation of poly--hydroxybutyric acid (PHB); in the course of 10–20 h (depending upon temperature) its content increased by 2.5–3.0 times. If, following darkening, the plants were once again exposed to light, an abrupt increase of nitrogen fixation intensity was observed and a simultaneous decrease of PHB content.It has been shown that lupin's exposure to light in¹⁴CO₂ atmosphere lasting 19 h resulted in the latter's incorporation into PHB, bacteroids and into the entire nodule; these processes developed almost in parallel.During the early period of vegetation growth prior to flowering, the PHB content of bacteroids decreased from 13–14 to 3–4% of dry weight, whereas the intensity of nitrogen fixation was raised. Concurrently increase of the activity of some enzymes connected with the PHB metabolism (acetoacetyl-CoA-reductase, acetyl-CoA-acetyl transferase, PHB-depolymerase, CoA-transferase of 3-ketoacids) occured. The plants' subsequent ageing and reduction of nitrogen fixation intensity led to a noticeable increase of PHB content and a decrease of the above mentioned enzymes' activity. The specific activity of \-hydroxybutyric dehydrogenase involved with PHB catabolism was high and was maintained at a constant level throughout the entire vegetative period.