Proteolytic Activity in Soybean Root Nodules : Activity in Host Cell Cytosol and Bacteroids throughout Physiological Development and Senescence

Root nodules were harvested from chamber-grown soybean (Glycine max L. Merrill cv Woodworth) plants throughout development. Apparent nitrogenase activity (acetylene reduction) peaked before seeds began to develop, but a significant amount of activity remained as the seeds matured. Nodule senescence was defined as the period in which residual nitrogenase activity was lost. During this time, soluble protein and leghemoglobin levels in the host cell cytosol decreased, and proteolytic activity against azocasein increased. Degradative changes were not detected in bacteroids during nodule senescence. Total soluble bacteroid protein per gram of nodule remained constant, and an increase in proteolytic activity in bacteroid extracts was not observed. These results are consistent with the view that soybean nodule bacteroids are capable of redifferentiation into free-living bacteria upon deterioration of the legume-rhizobia symbiosis.     

Inhibition of Nitrogen Fixation in Soybean Plants Supplied with Nitrate I. Nitrite Accumulation and Formation of Nitrosylleghemoglobin in Nodules

The accumulation of nitrite in nodules was investigated to elucidatethe mechanism of inhibition of nitrogen fixation in nodulesof soybean (Glycine max. [L.] Merr.) plants supplied with nitrate.Acetylene-reducing activity (ARA) in nodules fell within 24h as a result of the supply of exogenous nitrate, accompaniedby an increase in the accumulation of nitrite in the cytosolbut not in the bacteroids of nodules. Nitrate reductase (NR)activity in the nodule cytosol remained high, irrespective ofthe supply of nitrate. Nitrosylleghemoglobin (LbNO) was detectedspectrophotometrically in the extract from nodules in whichnitrogen fixation was inhibited by nitrate. In experiments invitro, it was found that LbNO was easily formed from leghemoglobinin the presence of nitrite and dithionite. Thus, it is suggested that nitrogen fixation was inhibited primarilyby a decrease in the function of leghemoglobin, attributableto the formation of LbNO, which was caused by the accumulationof nitrite generated from nitrate by NR in the nodule cytosol. (Received August 22, 1989; Accepted January 24, 1990)     

The effect of ammonium nitrate on the synthesis of nitrogenase and the concentration of leghemoglobin in pea root nodules induced by Rhizobium leguminosarum

The effects of NH4NO3 on the development of root nodules of Pisum sativum after infection with Rhizobium leguminosarum (strain PRE) and on the nitrogenase activity of the bacteroids in the nodule tissue were studied. The addition of NH4NO3 decreased the nitrogenase activity measured on intact nodules. This reduction of nitrogen fixation did not result from a reduced number of bacteroids or a decreased amount of bacteroid proteins per gram of nodule. The synthesis of nitrogenase, measured as the relative amount of incorporation of [35S]sulfate into the components I and II of nitrogenase was similarly not affected. The addition of NH4NO3 decreased the amount of leghemoglobin in the nodules and there was a quantitative correlation between the leghemoglobin content and the nitrogen-fixing capacity of the nodules. The conclusion is that the decrease of nitrogen-fixing capacity is caused by a decrease of the leghemoglobin content of the root nodules and not by repression of the nitrogenase synthesis.     

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.

     

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.     

Gas-exchange activity,carbohydrate status,and protein turnover in root nodule subpopulations of field pea (Pisum sativum L. cv. Century)

Root nodule ontogeny was followed in different parts of the root system of field peas (Pisum sativum L. cv. Century) to investigate the contribution to total nitrogen fixation by different nodule subpopulations. Seed-inoculated plants were grown to maturity in controlled-environment growth chambers. In a flow-through system nitrogenase activity (H₂-evolution in air) and nodulated-root respiration (net CO₂-evolution) were measured weekly or biweekly in different parts (top and mid) of the root system. Root nodule extracts were assayed for total soluble cytosolic protein, total heme, proteolytic capacity (at pH 7.0), soluble carbohydrates and starch. Total nitrogenase activity and nodule respiration were higher in the top zone, which was explained by differences in root and nodule mass. Nodule specific nitrogenase activity was similar in both zones, and gradually declined throughout the experiment. No differences were found between nodule subpopulations in the dry-matter specific concentrations of glucose, fructose, sucrose or starch. Neither did nodule concentrations of protein or leghemoglobin differ between the zones. Throughout reproductive growth, no decline was found in total or nodule specific nitrogenase activity, in any of the nodule subpopulations. Growth of the root nodules continued throughout the experiment, though growth of shoot and roots had ceased. The data gives no support for carbohydrate limitation in root nodules during pod-filling, since nodule respiration remained high, the concentration of soluble carbohydrates increased significantly, and the amount of starch was not reduced. We conclude that when this symbiosis is grown under controlled conditions, nitrogenase activity in nodules sub-sampled from the crown part of the root system is representative for the whole nodule population.     

Behaviour of nodulatedPisum sativum L. under short-term nitrate stress conditions

Nitrate (20 mM) applied to the root medium of 28-day-old nodulated pea plants (Pisum sativum L., cv. Jupiter) immediately retarded nodule growth and inhibited root nodulation. Acetylene-reducing and H₂-evolving nitrogenase activities were also significantly inhibited. The inhibitory effect of nitrate on nodule respiration was less pronounced while the respiration of roots was increased after the addition of nitrate. The levels of cytosol glutamine synthetase and nitrate reductase in nodule cytosol were permanently decreased from the 4th day after nitrate application. These results indicate that the inhibitory effect of high nitrate concentration on whole nodule metabolism is nonspecific in nature.     

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.     

Nitrate metabolism in soybean root nodules

The nitrate metabolism in nodules induced by Bradyrhizobium japonicum strain PJ17 on roots of soybean [ Glycine max (L.) Merr. cv. Hodgson] has been characterized by the nitrate reductase (NR; EC 1.6.6.1 and EC 1.6.6.3) activity of both partners of the symbiosis. NR activities of bacteroids and nodular cytosol were comparable and significantly higher than those of the roots. Nitrate reduction led to nitrite accumulation in root nodules, which was maximum after pod filling. The nodule had the capacity to metabolize nitrite via nitrite reductase (NiR; EC 1.6.6.4), at least in the cytosolic fraction. This activity was partly inhibited by the low content of free O₂ in the nodule. Indeed, nitrite accumulation decreased in the presence of an increased external pressure of O₂.     

Nitrate reduction and nitrogen fixation in symbiotic association Rhizobium-legumes

The inhibitory effect of nitrate on nitrogenase activity in root nodules of legume plants has been known for a long time. The major factor inducing changes in nitrogenase activity is the concentration of free oxygen inside nodules. Oxygen availability in the infected zone of nodule is limited, among others, by the gas diffusion resistance in nodule cortex. The presence of nitrate may cause changes in the resistance to O2 diffusion. The aim of this paper is to review literature data concerning the effect of nitrate on the symbiotic association between rhizobia and legume plants, with special emphasis on nitrogenase activity. Recent advances indicate that symbiotic associations of Rhizobium strains characterized by a high nitrate reductase activity are less susceptible to inhibition by nitrate. A thesis may be put forward that dissimilatory nitrate reduction, catalyzed by bacteroid nitrate reductase, significantly facilitates the symbiotic function of bacteroids.     

Effect of nitrate on the organic Acid and amino Acid composition of legume nodules

Nitrate supplied to legume plants inhibits the activity of nitrogenase in Rhizobium bacteroids in root nodules. The accumulation of amino N which is known to occur in Glycine max (L.) Merr. nodules as nitrogenase activity declines was studied in more detail by analysis of changes in free amino acid composition in response to high nitrate supply. A 6-fold increase in asparagine concentration in Bradyrhizobium japonicum bacteroids was found about the time of maximum nitrogenase inhibition. However, the accumulation of amino acids in soybean nodules lagged behind the inhibition of nitrogenase. Furthermore, in studies of a second legume, Phaseolus vulgaris (L.) inoculated with two different strains of Rhizobium phaseoli, a high nitrate treatment inhibited nitrogenase but had no significant effect on amino acid composition of nodules. The possibility that nitrate may interfere with the supply of carbon substrates to bacteroids was examined by the analysis of organic acids in legume nodules supplied with nitrate. Nitrate had a small (10-20%) negative effect on the concentration of tricarboxylic acid cycle acids in P. vulgaris nodules. However, in G. max nodules, high nitrate treatment resulted in significant increases in the concentration of malate, succinate, fumarate, and citrate. Thus, carbon deprivation of bacteroids also seems unlikely as a cause of the inhibition of nitrogenase by nitrate. There was a transient increase in ammonium concentration in P. vulgaris nodules in response to high nitrate treatment. This effect was rapid relative to other effects of nitrate on nodule composition and was roughly coincident with the rapid decline in acetylene reduction activity.     

Bacteroid distributions in alfalfa nodules upon dark-induced senescence and subsequent partial rejuvenation

Photosynthate availability directly controls the maturation, senescence and distribution of bacteroids (inoculum Rhizobium meliloti 102 F28) in alfalfa ( Medicago saliva L. cv. Buffalo) nodules. Mature, dinitrogen-fixing bacteroids were located principally in the middle section (region) of 6- to 8-week-old nodules in light-grown alfalfa plants. Upon dark treatment of the plants, bacteroids in the middle region of a nodule were induced to senescence while those in the tip region began to mature faster. Senescence and deterioration of bacteroids in the basal region of a nodule also were more advanced in the dark-treated plants. Sugar supplied exogenously during the dark period retarded the senescence process. Exposure of the dark-treated plants to light partially restored nitrogenase activity. The distribution of bacteroids in the rejuvenated nodules was similar to that of the light-grown plants.     

Nodule structure and functioning in Cicer arietinum as affected by nitrate

Chickpea (Cicer arietinum L.) cv. C-235 inoculated with Rhizobium sp. (Cicer) strain cv4Az was raised in sand culture under natural conditions with nitrogen-free nutrient solution. 45-d-old plants were treated with 20 and 50 mM KNO3 and sampling made 2 and 6 d after treatment. KNO3 application induced premature nodule senescence. Light microscopic investigations showed that KNO3 treatments resulted in structural degradation of the central bacteroidal tissue. The mass of green nodules increased by 35 % under these treatments. This was accompanied by a rapid decline in leghemoglobin (Lb) content of the nodules being 51 - 67 % lower than in control. The total soluble nodule proteins showed relatively minor changes under KNO3 treatments thus suggesting preferential degradation of Lb. These changes were associated with a rapid decline in N2-fixing activity. However, the decline in total soluble sugars was relatively minor as compared to acetylene reducing activity, thus indicating that sugar deprivation is not the cause of decreased nitrogen fixation ability. Glutathione reductase and ascorbate peroxidase activity showed a 10 - 20 % decrease in comparison with the control. Accumulation of H2O2 and structural degradation of the nodular tissue are considered to be the factors leading to nodule senescence under nitrate treatments. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effect of ammonium nitrate on the synthesis of nitrogenase and the concentration of leghemoglobin in pea root nodules induced by Rhizobium leguminosarum

The effects of NH₄NO₃ on the development of root nodules of Pisum sativum after infection with Rhizobium leguminosarum (strain PRE) and on the nitrogenase activity of the bacteriods in the nodule tissue were studied. The addition of NH₄NO₃ decreased the nitrogenase activity measured on intact nodules. This reduction of nitrogen fixation did not result from a reduced number of bacteroids or a decreased amount of bacteroid proteins per gram of nodule. The synthesis of nitrogenase, measured as the relative amount of incorporation of [³⁵S]sulfate into the components I and II of nitrogenase was similarly not affected.The addition of NH₄NO₃ decreased the amount of leghemoglobin in the nodules and there was a quantitative correlation between the leghemoglobin content and the nitrogen-fixing capacity of the nodules. The conclusion is that the decrease of nitrogen-fixing capacity is caused by a decrease of the leghemoglobin content of the root nodules and not by repression of the nitrogenase synthesis.     

Studies on soybean nodule senescence

Soybean Glycine max. L. Merr. nodule senescence was studied using the loss of acetylene reduction by intact tap root nodules as its indication. Tap root nodules from two varieties (Calland and Beeson) of field-grown soybeans were used. The specific activities of nitrogenase (micromoles/minute gram fresh weight of nodules) as measured by the acetylene reduction assay decreased abruptly between 58 to 65 and 68 to 75 days after planting the Beeson and Calland soybeans, respectively. Major changes were not detected in dry weight, total nitrogen, and leghemoglobin levels during the period when in vivo nitrogenase activity declined. Ammonium levels in the cytosol of nodules and poly-β-hydroxybutyrate increased moderately just prior to or coincidental with the loss of nitrogenase activity. Neither enzymes that have been postulated to be involved in ammonium assimilation nor NADP⁺-specific isocitrate dehydrogenase exhibited any large changes in specific activities during the initial period when nitrogenase activity declined.     

Sensitive fluorometric assay for leghemoglobin

A sensitive spectrofluorometric assay for leghemoglobin is based upon the action of hot saturated oxalic acid on heme proteins. The assay will detect 200 ng of leghemoglobin per milliliter and is specific enough to permit estimation in single nodules or extracts of whole roots. The leghemoglobin concentration measured fluorometrically shows a correlation with nitrogenase [C₂H₂] activity, even during nodule senescence, when standard colorimetric assays may overestimate leghemoglobin.     

Some Enzymes of Hydrogen Peroxide Metabolism in Leaves and Root Nodules of Medicago sativa

Leaves and nodules (bacteroids and cytosol) of alfalfa (Medicago sativa L. cv Aragon) plants inoculated with Rhizobium meliloti strain 102F51 have been analyzed for the presence of the enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (EC 1.11.1.6), and peroxidase (EC 1.11.1.7). All three fractions investigated (leaves, bacteroids, and nodular cytosol) show Cu,Zn-SOD activity. Besides, the bacteroids and cytosol of nodules possess CN⁻-insensitive SOD activities. Studies of SOD inactivation with H₂O₂ indicate that, very likely, a Mn-SOD is present in the bacteroids, and suggest that the cytosol contain both Mn-SOD and Fe-SOD. Bacteroids show high catalase activity but lack peroxidase. By contrast, the nodule cytosol exhibits an elevated peroxidase activity as compared with the foliar tissue; this activity was completely inhibited by 50 to 100 micromolar KCN. The significantly lower contents of H₂O₂ and malondialdehyde (a product of lipid peroxidation) in nodules with respect to those in leaves reveal that the above-mentioned bacteroid and cytosol enzymes act in an efficient and combined manner to preserve integrity of nodule cell membranes and to keep leghemoglobin active.     

Inhibition of Nitrogen Fixation in Soybean Plants Supplied with Nitrate II. Accumulation and Properties of Nitrosylleghemoglobin in Nodules

The accumulation of nitrosylleghemoglobin (LbNO) in nodulesand the properties of LbNO in vitro were investigated in connectionwith the inhibition of nitrogen fixation in soybean nodulesby nitrate. The leghemoglobin extracted under argon gas from nodules ofplants supplied with nitrate consisted mainly of LbNO, as judgedfrom the spectrum which corresponded to that of LbNO formedin vitro by the reaction of leghemoglobin with nitrite in thepresence of dithionite or by the combination of ferrous leghemoglobin(Lb²⁺) with nitric oxide. Further, LbNO formed in vivo was easilydissociated by visible light, as was LbNO formed in vitro. Thus,authentic LbNO does actually accumulate in nodules. Most of the leghemoglobin was of the ferrous type in nodulesof plants supplied with nitrate. Some LbNO appeared to be derivedfrom LbO₂ which was deoxygenated by nitrite. The increase inlevels of LbNO in nodules paralleled the decrease in acetylenereducing activity. These results indicate that the decrease in nitrogenase activityin nodules of soybean plants supplemented with nitrate is causedby the decrease in levels of LbO₂ that carries oxygen into bacteroids,which results from the formation of LbNO (Received August 22, 1989; Accepted December 4, 1989)     

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.