Ammonia (C-Methylamine) Transport across the Bacteroid and Peribacteroid Membranes of Soybean Root Nodules

[¹⁴C]Methylamine (MA; an analog of ammonia) was used to investigate ammonia transport across the bacteroid and peribacteroid membranes (PBM) from soybean (Glycine max) root nodules. Free-living Bradyrhizobium japonicum USDA110 grown under nitrogen-limited conditions showed rapid MA uptake with saturation kinetics at neutral pH, indicative of a carrier. Exchange of accumulated MA for added ammonia occurred, showing that the carrier recognized both NH₄⁺ and CH₃NH₃⁺. MA uptake by isolated bacteroids, on the other hand, was very slow at low concentrations of MA and increased linearly with increasing MA concentration up to 1 millimolar. Ammonia did not inhibit MA by isolated bacteroids and did not cause efflux of accumulated MA. PBM-enclosed bacteroids (peribacteroid units [PBUs]) were qualitatively similar to free bacteroids with respect to MA transport. The rates of uptake and efflux of MA by PBUs were linearly dependent on the imposed concentration gradient and unaffected by NH₄Cl. MA uptake by PBUs increased exponentially with increasing pH, confirming that the rate increased linearly with increasing CH₃NH₂ concentration. The results are consistent with other evidence that transfer of ammonia from the nitrogen-fixing bacteroid to the host cytosol in soybean root nodules occurs solely by simple diffusion of NH₃ across both the bacteroid and peribacteroid membranes.     

Morphological and structural adaptation of nodules of cowpea to functioning under sub- and supra-ambient oxygen pressure

Nodules of cowpea (Vigna unguiculata (L.) Walp. cv. Vita 3:Bradyrhizobium CB 756) from 28-d-old plants cultured for 23 d with their root systems maintained in O₂ levels from 1 to 80% (v/v, in N₂) in the external gas phase showed a range of structural changes which have been interpreted in relation to an over- or under-supply of O₂. A response to the partial pressure of O₂ in the gas phase (pO₂) was noted with respect to nodule size, lenticel development, the relative distributions of cortical and infected central tissue, the differentiation of cortex, especially the inner cortex, the frequency and size of infected and uninfected interstitial cells, the volume of extracellular spaces both in cortex and infected tissue, and in the frequency of bacteroids. As a consequence of these changes the surface area of inner cortex relative to the nitrogenase-containing units of fixing tissue (infected cells or bacteroids) was increased by as much as 20-fold. Effectiveness of bacteroid functioning increased from 0.10 ± 0.02 · 10^-9 μmol acetylene reduced per bacteroid in air-grown nodules to 0.9 ± 0.16 · 10^-9 (same units) per bacteroid in those cultured in 1% O₂. This work was supported by a grant from the Australian Research Council (to C.A.A.) and an Australian International Development Assistance Bureau postgraduate fellowship (to F.D.D.). The authors wish to thank Dr. W.F.C. Blumer for his considerable help with morphometric analysis, Dr. J. Kuo for guidance in the use of histological techniques, and to Dr. J.S. Pate for the suggestion that lenticel development might be quantified by surface staining of nodules.     

Decreased Exopolysaccharide Synthesis by Anaerobic and Symbiotic Cells of Bradyrhizobium japonicum

Experiments were conducted to determine whether symbiotic bacteroids of Bradyrhizobium japonicum produce exopolysaccharide within soybean (Glycine max [L.] Merr. cv `Lee 74') nodules. B. japonicum strains RT2, a derivative of USDA 110 with resistance to streptomycin and rifampicin, and RT176-1, a mutant deficient in exopolysaccharide synthesis, were used. Although aerobically cultured RT2 produced 1550 micrograms of exopolysaccharide per 10¹⁰ cells, root nodules formed by RT2 contained only 55.7 micrograms of polysaccharide per 10¹⁰ bacteroids, indicating that little exopolysaccharide synthesis occurred within the nodules. The polysaccharide level of RT2 nodules was about equal to that of nodules containing the exopolysaccharide mutant RT176-1 (61.0 micrograms per 10¹⁰ bacteroids). Gas chromatographic analysis showed that the sugar composition of polysaccharide from nodules of RT2 or RT176-1 was almost the same as that of polysaccharide from unnodulated root tissue, but differed strikingly from that of rhizobial exopolysaccharide from aerobic cultures. Thus, the host plant and not the bacteroids was probably the source of most or all of the polysaccharide in the nodule extracts. Also, bacteroids from nodules failed to bind soybean lectin, confirming the absence of an exopolysaccharide capsule.     

Nitrate reductase activity of free-living and symbiotic uptake hydrogenase-positive and uptake hydrogenase-negative strains of Bradyrhizobium japonicum

The nitrate reductase activity (NR) of selected uptake hydrogenase-positive (hup ⁺) and uptake hydrogenase-negative (hup ^-) strains of Bradyrhizobium japonicum were examined both in free-living cells and in symbioses with Glycine max L. (Marr.) cv. Williams. Bacteria were cultured in a defined medium containing either 10 mM glutamate or nitrate as the sole nitrogen source. Nodules and bacteriods were isolated from plants that were only N₂-dependent or grown in the presence of 2 mM KNO₃. Rates of activity in nodules were determined by an in vivo assay, and those of cultured cells and bacteriods were assayed after permeabilization of the cells with alkyltrimethyl ammonium bromide. All seven strains examined expressed NR activity as free-living cells and as symbiotic forms, regardless of the hup genotype of the strain used for inoculation. Although the presence of nitrate increased nitrate reduction by cultures cells and nodules, no differences in NR activity were observed between bacteroids isolated from nodules of plants fed with nitrate or grown on N₂-fixation exclusively. Cultured cells, nodules and bacteriods of strains with hup ^- genotype (USDA 138, L-236, 3. 15B3 and PJ17) had higher rates of NR activity than those with hup ⁺ genotype (USDA 110, USDA 122 DES and CB1003). These results suggest that NR activity is reduced in the presence of a genetic determinant associated with the hup region of B. japonicum.Abbreviations EDTA ethylene-diamine tetraacetic acid - Hup hydrogen uptake - MOPS 3-(N-morpholino)-propane sulfonic acid - NR nitrate reductase - PVP polyvinyl-polypyrrolidone - Tris Tris(hydroxymethyl)-aminomethane     

Bacteroid-encoded proteins are secreted into the peribacteroid space by Rhizobium leguminosarum

Bacteroids of Rhizobium leguminosarum in root nodules of Pisum sativum are enclosed by a plant-derived peribacteriod membrane (PBM). The contents of the interstitial peribacteroid space (PBS) between bacteroid membrane and PBM were isolated by a controlled osmotic shock of PBM-enclosed bacteroids and analysed by two-dimensional gel electrophoresis. Silver staining revealed approximately 40 PBS polypeptides. Ex planta ³⁵S-methionine labeling of PBM-enclosed bacteroids revealed that about 90% of the PBS proteins are synthesized by the bacteroid. Approximately 30% of the PBS polypeptides are common between the PBS and the periplasmic space of free-living bacteria; one (38kDa) PBS protein is also excreted by free-living bacteria in the bacterial culture medium. At least four bacteroid-encoded PBS polypeptides were clearly identified as symbiosis-specific.     

Differentiation of nodules of Glycine max

Plants of Glycine max var. Caloria, infected as 14 d old seedlings with a defined titre of Rhizobium japonicum 3Il b85 in a 10 min inoculation test, develop a sharp maximum of nitrogenase activity between 17 and 25 d after infection. This maximum (14±3 nmol C₂H₄ h^-1 mg nodule fresh weight^-1), expressed as per mg nodule or per plant is followed by a 15 d period of reduced nitrogen fixation (20–30% of peak activity). 11 d after infection the first bacteroids develop as single cells inside infection vacuoles in the plant cells, close to the cell wall and infection threads. As a cytological marker for peak multiplication of bacteroids and for peak N₂-fixation a few days later the association of a special type of nodule mitochondria with amyloplasts is described. 20 d after inoculation, more than 80% of the volume of infected plant cells is occupied by infection vacuoles, mostly containing only one bacteroid. The storage of poly--hydroxybutyrate starts to accumulate at both ends of the bacteroids. Non infected plant cells are squeezed between infected cells (25d), with infection vacuoles containing now more than two (up to five) bacteroids per section. Bacteroid development including a membrane envelope is also observed in the intercellular space between plant cells. 35 d after infection, more than 50% of the bacteroid volume is occupied by poly--hydroxybutyrate. The ultrastructural differentiation is discussed in relation to some enzymatic data in bacteroids and plant cell cytoplasm during nodule development.     

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     

Evidence for a Third Uptake Hydrogenase Phenotype among the Soybean Bradyrhizobia

The existence of a hydrogen uptake host-regulated (Hup-hr) phenotype was established among the soybean bradyrhizobia. The Hup-hr phenotype is characterized by the expression of uptake hydrogenase activity in symbiosis with cowpea but not soybean. Uptake hydrogenase induction is not possible under free-living cultural conditions by using techniques developed for uptake hydrogenase-positive (Hup⁺) Bradyrhizobium japonicum. Hydrogen oxidation by Hup-hr phenotype USDA 61 in cowpea symbioses was significant because hydrogen evolution from nitrogen-fixing nodules was not detected. An examination for uptake hydrogenase activity in soybean and cowpea with 123 strains diverse in origin and serology identified 16 Hup⁺ and 28 Hup-hr phenotype strains; the remainder appeared to be Hup⁻. The Hup-hr phenotype was associated with serogroups 31, 76, and 94, while strains belonging to serogroups 6, 31, 110, 122, 123, and 38/115 were Hup⁺. Hup⁺ strains of the 123 serogroup typed positive with USDA 129-specific antiserum. The presence of the uptake hydrogenase protein in cowpea bacteroids of Hup⁺ strains was demonstrated with immunoblot analyses by using antibodies against the 65-kDa subunit of uptake hydrogenase purified from strain SR470. However, the hydrogenase protein of Hup-hr strains was not detected. Results of Southern hybridization analyses with pHU1 showed the region of DNA with hydrogenase genes among Hup⁺ strains to be similar. Hybridization was also obtained with Hup-hr strains by using a variety of cloned DNA as probes including hydrogenase structural genes. Both hydrogenase structural genes also hybridized with the DNA of four Hup⁻ strains.     

Variability in molybdenum uptake activity in Bradyrhizobium japonicum strains.

Twenty naturally occurring strains of Bradyrhizobium japonicum in 11 serogroups were screened for the ability to take up Mo as bacteroids from soybean root nodules. The strains varied greatly in their ability to take up Mo in a 1-min period. The best strain was USDA 136, which had an Mo uptake activity of almost 3.0 pmol/min per mg of bacteroid (dry weight). In contrast, the poorest strain, USDA 62, had an Mo uptake activity of 0.35 pmol of Mo per min per mg of bacteroid. There were similarities in Mo uptake ability among most of the same serogroup members. The variability in Mo uptake rates between the best (USDA 136 and USDA 122) and poorest (USDA 62 and USDA 140) strains was attributed to their differing affinities for Mo. Double-reciprocal plots of velocity versus substrate indicated a Km for USDA 136 and USDA 122 of 0.045 and 0.054 microM, respectively, whereas strains USDA 62 and USDA 140 both exhibited an apparent Km for MoO42- of about 0.36 microM. The two strains with the higher-affinity Mo binding also accumulated four to five times as much Mo over a 30-min period as the other strains. Soybeans were grown in Mo-deficient and Mo-supplemented conditions after inoculation with the three top-ranking Mo uptake strains and the three poorest Mo uptake strains. Two separate greenhouse studies indicated that Mo supplementation significantly increased the N2 fixation activity of USDA 140 nodules; up to a 35% increase in specific nitrogen fixation activity of nodules due to Mo supplementation was observed. Strain USDA 62 nodule N2 fixation responded positively to Mo supplementation in one of the two experiments. The results indicate that MoO42- transport and, specifically, affinity for Mo by the bacteroid may ultimately affect symbiotic N2 fixation activity. Attempts to reactivate nitrogenase by adding molybdate to bacteroids from plants grown in Mo-deficient conditions were unsuccessful.     

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.

     

Labeling of Carbon Pools in Bradyrhizobium japonicum and Rhizobium leguminosarum bv viciae Bacteroids following Incubation of Intact Nodules with CO(2)

The aim of the work reported here was to ascertain that the patterns of labeling seen in isolated bacteroids also occurred in bacteroids in intact nodules and to observe early metabolic events following exposure of intact nodules to ¹⁴CO₂. Intact nodules of soybean (Glycine max L. Merr. cv Ripley) inoculated with Bradyrhizobium japonicum USDA 110 and pea (Pisum sativum L. cv Progress 9) inoculated with Rhizobium leguminosarum bv viciae isolate 128C53 were detached and immediately fed ¹⁴CO₂ for 1 to 6 min. Bacteroids were purified from these nodules in 5 to 7 min after the feeding period. In the cytosol from both soybean and pea nodules, malate had the highest radioactivity, followed by citrate and aspartate. In peas, asparagine labeling equaled that of aspartate. In B. japonicum bacteroids, malate was the most rapidly labeled compound, and the rate of glutamate labeling was 67% of the rate of malate labeling. Aspartate and alanine were the next most rapidly labeled compounds. R. leguminosarum bacteroids had very low amounts of ¹⁴C and, after a 1-min feeding, malate contained 90% of the radioactivity in the organic acid fraction. Only a trace of activity was found in aspartate, whereas the rate of glutamate and alanine labeling approached that of malate after 6 min of feeding. Under the conditions studied, malate was the major form of labeled carbon supplied to both types of bacteroids. These results with intact nodules confirm our earlier results with isolated bacteroids, which showed that a significant proportion of provided labeled substrate, such as malate, is diverted to glutamate. This supports the conclusion that microaerobic conditions in nodules influence carbon metabolism in bacteroids.     

Reproductive capacity of bacteroids in nodules of Trifolium repens L. and Glycine max (L.) Merr.

Reproductive growth of intracellular bacteria from isolated protoplasts in nodules of clover and soybean was directly investigated using a microchamber with visual and video recording. Differentiated bacteriods from clover nodules uniformly failed to reproduce. Such growth as occurred came from undifferentiated rhizobia from within the protoplast or extracellularly in the nodule. Plating investigation gave results in agreement with this conclusion. Osmoprotective media failed to secure the reproduction of differentiated clover bacteroids. Reproductive growth of bacteroids from protoplasts and crushed nodules of soybean was regularly observed in the microchamber and determined as proportionate colony-forming ability (CFA) on laboratory media. The CFA markedly increased with age of nodule and with the addition of nodule or root extract. The promoting effect of such extracts was reduced after heating for 60 min at 100°C, and lost completely after 20 min at 121°C. High osmolarity in the suspending and culture media was detrimental to bacteroid recovery.Abbreviations BMM Bergersen's modified medium - B^+m BMM with additional mannitol - CDB Chlamydomonas dilution buffer - PDB protoplast dilution buffer - PDB PDB without mannitol or sorbitol - RMM Rhizobium minimal medium - R^+m RMM with mannitol instead of sucrose - YMA, YMB yeast mannitol agar and broth, respectively. For details, see Materials and methods     

Metabolism of C-labeled photosynthate and distribution of enzymes of glucose metabolism in soybean nodules

The metabolism of translocated photosynthate by soybean (Glycine max L. Merr.) nodules was investigated by ¹⁴CO₂-labeling studies and analysis of nodule enzymes. Plants were exposed to ¹⁴CO₂ for 30 minutes, followed by ¹²CO₂ for up to 5 hours. The largest amount of radioactivity in nodules was recovered in neutral sugars at all sampling times. The organic acid fraction of the cytosol was labeled rapidly. Although cyclitols and malonate were found in high concentrations in the nodules, they accumulated less than 10% of the radioactivity in the neutral and acidic fractions, respectively. Phosphate esters were found to contain very low levels of total label, which prohibited analysis of the radioactivity in individual compounds. The whole nodule-labeling patterns suggested the utilization of photosynthate for the generation of organic acids (principally malate) and amino acids (principally glutamate).

The radioactivity in bacteroids as a percentage of total nodule label increased slightly with time, while the percentage in the cytosol fraction declined. The labeling patterns for the cytosol were essentially the same as whole nodule-labeling patterns, and they suggest a degradation of carbohydrates for the production of organic acids and amino acids. When it was found that most of the radioactivity in bacteroids was in sugars, the enzymes of glucose metabolism were surveyed. Bacteroids from nodules formed by Rhizobium japonicum strain 110 or strain 138 lacked activity for phosphofructokinase and NADP-dependent 6-phosphogluconate dehydrogenase, key enzymes of glycolysis and the oxidative pentose-phosphate pathways. Enzymes of the glycolytic and pentose phosphate pathways were found in the cytosol fraction.

In three experiments, bacteroids contained about 10 to 30% of the total radioactivity in nodules 2 to 5 hours after pulse-labeling of plants, and 60 to 65% of the radioactivity in bacteroids was in the neutral sugar fraction at all sampling times. This strongly suggests some absorption and metabolism of sugars by bacteroids in spite of the lack of key enzymes. Bacteroids did possess enzymes for the formation of hexose phosphates from glucose or fructose. Radioactivity in α,α-trehalose in bacteroids increased until, after 5 hours, trehalose was a major labeled compound in bacteroids. Thus, trehalose synthesis may be a major fate of sugars entering bacteroids.

     

Soybean response to nodulation by bradyrhizobia differing in rhizobitoxine phenotype

Rhizobitoxine-producing (RT⁺) strains of Bradyrhizobium japonicum, differing in their abilities to induce foliar chlorosis with ‘Forrest’ soybean (Glycine max [L.] Merr.), were evaluated for effects on short term shoot productivity, nodulation, N₂ fixation, and nodule protein production under greenhouse conditions. Soybeans were singly inoculated with washed suspensions of (Group II) USDA strains 31, 46, 76, 94, 110, 123 or 130. Strains USDA 110 and USDA 123 (Group I/Ia) were included as RT-controls. The plants were cultured in the absence of combined N in horticultural-grade vermiculite for 49 days. Beginning 21 days after planting, plants were evaluated weekly for chlorophyll, leaf protein and biomass accumulation, nodular contents of leghemoglobin, soluble protein and RT, and total shoot N content. Rhizobitoxine was detected in nodules of all RT⁺ strains with the exception of USDA 31. However, only USDA 76 and USDA 94 produced both quantifiable concentrations of RT and symptoms of RT-induced chlorosis. Coincident with moderate to severe chlorosis were reductions in chlorophyll concentrations, shoot and nodule dry weight, leaf protein and total N₂ fixation. During extended periods of severe chlorosis, reductions in Lb and soluble nodular protein were observed. Based on carbon accumulation, all non-chlorotic treatments were statistically more productive than the chlorotic treatments. Similarly, non-chlorotic Group II treatments tended to fix less carbon relative to the RT-Group I/Ia controls, although these differences were not statistically significant. The results of this study suggest that, in the absence of discernable foliar chlorosis, the effect of RT⁺ (Group II) nodulation on short term soybean productivity is minimal. Published as Miscellaneous Paper No. 1439 of the Delaware Agricultural Experiment Station. Published as Miscellaneous Paper No. 1439 of the Delaware Agricultural Experiment Station.     

Identifying abnormalities in symbiotic development between Trifolium spp. and Rhizobium leguminosarum bv. trifolii leading to sub-optimal and ineffective nodule phenotypes

Background and Aims

Legumes overcome nitrogen limitations by entering into a mutualistic symbiosis with N₂-fixing bacteria (rhizobia). Fully compatible associations (effective) between Trifolium spp. and Rhizobium leguminosarum bv. trifolii result from successful recognition of symbiotic partners in the rhizosphere, root hair infection and the formation of nodules where N₂-fixing bacteroids reside. Poorly compatible associations can result in root nodule formation with minimal (sub-optimal) or no (ineffective) N₂-fixation. Despite the abundance and persistence of strains in agricultural soils which are poorly compatible with the commercially grown clover species, little is known of how and why they fail symbiotically. The aims of this research were to determine the morphological aberrations occurring in sub-optimal and ineffective clover nodules and to determine whether reduced bacteroid numbers or reduced N₂-fixing activity is the main cause for the Sub-optimal phenotype.

Methods

Symbiotic effectiveness of four Trifolium hosts with each of four R. leguminosarum bv. trifolii strains was assessed by analysis of plant yields and nitrogen content; nodule yields, abundance, morphology and internal structure; and bacteroid cytology, quantity and activity.

Key Results

Effective nodules (Nodule Function 83–100 %) contained four developmental zones and N₂-fixing bacteroids. In contrast, Sub-optimal nodules of the same age (Nodule Function 24–57 %) carried prematurely senescing bacteroids and a small bacteroid pool resulting in reduced shoot N. Ineffective-differentiated nodules carried bacteroids aborted at stage 2 or 3 in differentiation. In contrast, bacteroids were not observed in Ineffective-vegetative nodules despite the presence of bacteria within infection threads.

Conclusions

Three major responses to N₂-fixation incompatibility between Trifolium spp. and R. l. trifolii strains were found: failed bacterial endocytosis from infection threads into plant cortical cells, bacteroid differentiation aborted prematurely, and a reduced pool of functional bacteroids which underwent premature senescence. We discuss possible underlying genetic causes of these developmental abnormalities and consider impacts on N₂-fixation of clovers.     

Lysis of bacterioids in the vicinity of the host cell nucleus in an ineffective (fix-) root nodule of soybean (Glycine max)

In nodules of Glycine max cv. Mandarin infected with a nod ⁺fix^- mutant of Rhizobium japonicum (RH 31-Marburg), lysis of bacteroids was observed 20 d after infection, but occurred in the region around the host cell nucleus, where lytic compartments were formed. Bacteroids, and peribacteroid membranes in other parts of the host cell remained stable until senescence (40d after infection). With two other nod⁺ fix^- mutants of R. japonicum either stable bacteroids and peribacteroid membranes were observed throughout the cell (strain 61-A-165) or a rapid degeneration of bacteroids without an apparent lysis (strain USDA 24) occurred. The size distribution of RH 31-Marburg-infected nodules exhibited only two maxima compared with four in wild-type nodules and nodule leghaemoglobin content was found to be reduced to about one half that of the wild type. The RH 31-Marburg-nodule type is discussed in relation to the stability of the bacteroids and the peribacteroid membrane system in soybean.     

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.     

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 nutrition and the development and senescence of nodules on cowpea seedlings

Cowpea (Vigna unguiculata (L.) Walp cv. Vita 3) seedlings inoculated with Rhizobium strain CB756 were cultured with their root systems maintained in air or in Ar: O₂ (80:20, v/v) during early nodule development (up to 24 d after sowing). Compared with those in air, seedlings in Ar:O₂ showed progressive N deficiency with inhibited shoot growth, reduced ribulose-1,5-bisphosphate carboxylase and total protein levels and loss of chlorophyll in the leaves. Nodule initiation, differentiation of infected and uninfected nodule tissues and the ultrastructure of bacteriod-containing cells were similar in the air and Ar: O₂ treatments up to 16 d after sowing. Thereafter the Ar: O₂ treatment caused cessation of growth and development of nodules, reduced protein levels in bacteroids and nodule plant cells, and progressive degeneration of nodule ultrastructure leading to premature senescence of these organs. Provision of NO ₃ ^- (0.1–0.2 mM) to Ar: O₂-grown seedlings overcame the abovementioned consequences of N₂ deficiency on nodule and plant growth, but merely delayed the degenerative effects of Ar: O₂ treatment on nodule structure and senescence. Treatment of Ar: O₂-grown seedlings with NO ₃ ^- greatly increased the protein level of nodules but the increase was largely restricted to the plant cell fraction as opposed to the bacteroids. By contrast, NO ₃ ^- treatment of air-grown seedlings increased protein of bacteroid and host nodule fractions to the same relative extents when compared with air-grown plants not supplemented with NO ₃ ^- . These findings, taken together with studies of the distribution of N in nodules of symbiotically effective plants grown from ¹⁵N-labeled seed, indicate that direct incorporation of fixation products by bacteroids may be a critical feature in the establishment and continued growth of an effective symbiosis in the cowpea seedling.Abbreviation RuBPCase ribulose-1,5-bisphosphate carboxylase     

Composition and Distribution of Adenylates in Soybean (Glycine max L.) Nodule Tissue

Adenylates (ATP, ADP, and AMP) may play a central role in the regulation of the O2-limited C and N metabolism of soybean nodules. To be able to interpret measurements of adenylate levels in whole nodules and to appreciate the significance of observed changes in adenylates associated with changes in O2-limited metabolism, methods were developed for measuring in vivo levels of adenylate pools in the cortex, plant central zone, and bacteroid fractions of soybean (Glycine max L. Merr cv Maple Arrow x Bradyrhizobium japonicum strain USDA 16) nodules. Intact nodulated roots were either frozen in situ by flushing with prechilled Freon-113(-156[deg]C) or by rapidly (<1 s) uprooting plants and plunging them into liquid N2. The adenylate energy charge (AEC = [ATP + 0.5 x ADP]/[ATP + ADP + AMP]) of whole-nodule tissue (0.65 [plus or minus] 0.01, n = 4) was low compared to that of subtending roots (0.80 [plus or minus] 0.03, n = 4), a finding indicative of hypoxic metabolism in nodules. The cortex and central zone tissues were dissected apart in lyophilized nodules, and AEC values were 0.84 [plus or minus] 0.04 and 0.61 [plus or minus] 0.03, respectively. Although the total adenylate pool in the lyophilized nodules was only 41% of that measured in hydrated tissues, the AEC values were similar, and the lyophilized nodules were assumed to provide useful material for assessing adenylate distribution. The nodule cortex contained 4.4% of whole-nodule adenylates, with 95.6% being located in the central zone. Aqueous fractionation of bacteroids from the plant fraction of whole nodules and the use of marker enzymes or compounds to correct for recovery of bacteroids and cross-contamination of the bacteroid and plant fractions resulted in estimates that 36.2% of the total adenylate pool was in bacteroids, and 59.4% was in the plant fraction of the central zone. These are the first quantitative assessments of adenylate distribution in the plant and bacteroid fractions of legume nodules. These estimates were combined with theoretical calculations of rates of ATP consumption in the cortex (9.5 nmol g-1 fresh weight of nodule s-1), plant central zone (38 nmol g-1 fresh weight of nodule s-1), and bacteroids (62 nmol g-1 fresh weight of nodule s-1) of soybean nodules to estimate the time constants for turnover of the total adenylate pool and the ATP pool within each nodule fraction. The low values for time constant (1.6-5.8 s for total adenylate, 0.9-2.5 s for ATP only) in each fraction reflect the high metabolic activity of soybean nodules and provide a background for further studies of the role of adenylates in O2-limited nodule metabolism.