Inhibition of ornithine decarboxylase activity reduces polyamine levels and growth ofAgrobacterium tumefaciens

Flavins in different compartments of effective nodules fromGlycine max cv Maple Arrow xBradyrhizobium japonicum strains were studied by spectrophotometry and chromatographic techniques. Flavins in the peribacteroid space were riboflavin (80%) and FMN (20%), as identified by TLC and HPLC. Flavin concentrations in the soybean root nodule cytoplasm, in the symbiosome space (PBS) and in the cytosol of bacteroids were monitored between 20 and 40 days post infection (d.p.i.) Between the 20th and 29th d.p.i. an at least four times higher flavin/protein ratio was found in PBS of effective nodules compared with the nodule cytoplasm. Between nitrogenase activity in the free-living state and bacterial flavin accumulation, no correlation could be observed. Flavin accumulation is not restricted to an effective symbiosis, as indicated by the analysis of ineffective nodules with strainB. japonicum RH-31 Marburg. Flavin accumulation is absent in uninfected soybean root tissue and in free-living rhizobia, thus indicating that flavin accumulation is a result of symbiotic interaction. Flavin accumulation is also missing in nodules with a hypersensitive response against the bacteria.     

Rhizobium trifolii mutants deficient in exopolysaccharide production

Spontaneous mutants of Rhizobium trifolii 24AR5 which did not produce exopoly-saccharide were isolated. The non-mucoid mutants formed small white and ineffective nodules on both red and white clover. These nodules contained infection threads, but only a small number of bacteria were released into nodule cells, and bacteroids were rarely observed. The non-mucoid phenotype was not complemented by the symbiotic plasmid (pJB5JI) of Rhizobium leguminosarum.     

Isolation and symbiotic characterization of transposon Tn5-induced arginine auxotrophs of Sinorhizobium meliloti

Seventeen arginine auxotrophic mutants of Sinorhizobium meliloti Rmd201 were isolated by random transposon Tn5 mutagenesis using Tn5 delivery vector pGS9. Based on intermediate feeding studies, these mutants were designated as argA/argB/argC/argD/argE (ornithine auxotrophs), argF/argI, argG and argH mutants. The ornithine auxotrophs induced ineffective nodules whereas all other arginine auxotrophs induced fully effective nodules on alfalfa plants. In comparison to the parental strain induced nodule, only a few nodule cells infected with rhizobia were seen in the nitrogen fixation zone of the nodule induced by the ornithine auxotroph. TEM studies showed that the bacteroids in the nitrogen fixation zone of ornithine auxotroph induced nodule were mostly spherical or oval unlike the elongated bacteroids in the nitrogen fixation zone of the parental strain induced nodule. These results indicate that ornithine or an intermediate of ornithine biosynthesis, or a chemical factor derived from one of these compounds is required for the normal development of nitrogen fixation zone and transformation of rhizobial bacteria into bacteroids during symbiosis of S. meliloti with alfalfa plants.     

Isolation and symbiotic characterization of aromatic amino acid auxotrophs of Sinorhizobium meliloti

Ten aromatic amino acid auxotrophs of Sinorhizobium meliloti (previously called Rhizobium meliloti) Rmd201 were generated by random mutagenesis with transposon Tn5 and their symbiotic properties were studied. Normal symbiotic activity, as indicated by morphological features, was observed in the tryptophan synthase mutants and the lone tyrosine mutant. The trpE and aro mutants fixed trace amounts of nitrogen whereas the phe mutant was completely ineffective in nitrogen fixation. Histology of the nodules induced by trpE and aro mutants exhibited striking similarities. Each of these nodules contained an extended infection zone and a poorly developed nitrogen fixation zone. Transmission electron microscopic studies revealed that the bacteroids in the extended infection zone of these nodules did not show maturation tendency. A leaky mutant, which has a mutation in trpC, trpD, or trpF gene, was partially effective in nitrogen fixation. The histology of the nodules induced by this strain was like that of the nodules induced by the parental strain but the inoculated plants were stunted. These studies demonstrated the involvement of anthranilic acid and at least one more intermediate of tryptophan biosynthetic pathway in bacteroidal maturation and nitrogen fixation in S. meliloti. The alfalfa plant host seems to provide tryptophan and tyrosine but not phenylalanine to bacteroids in nodules.     

Effects of some surfactant fungicides on Rhizobium trtfolii and its symbiotic relationship with white clover

Effects of the surfactant apple mildew eradicants Manoxol OT, Alk 3, Triton X45, PP 222, Ethylan CP, Hyamine 3500, Aromox DMMCDW and Aromox C12W on growth and respiration of R. trifolii in vitro and on its symbiotic nitrogen fixation in association with white clover were measured. Most treatments reduced growth up to 8 wk; after 12 wk plant weight and nitrogenase activity were significantly reduced (P = 0.01) by Triton X45, Manoxol OT and PP 222 at concentrations of 1000, 2500 and 5000 μg/gram of soil respectively. Electron microscopy showed that bacteroids in root nodules where nitrogen fixation was inhibited had contracted away from the host membrane leaving an intracellular space. Damage to the bacteroid membrane was apparent. Surfactants had no demonstrable effect on adhesion of the bacteria to root hairs. In apple orchards run-off of surfactant fungicides, applied to apple trees at the recommended rates is unlikely to have adverse effects on Rhizobium nodulated white clover.     

The origin of the membrane envelope surrounding the bacteria and bacteroids and the presence of glycogen in clover root nodules

Summary Thin sections of clover nodules were examined by electron microscopy. The emergence of the bacteria from the infection thread was found to occur by a process of phagocytosis. No evidence was found, using glutar-aldehyde-osmium as a fixative, that there is any connection between the membrane envelope and the endoplasmic reticulum.Electron dense granules, previously observed in clover nodule bacteria, were identified as glycogen granules. These glycogen granules accumulated in bacteria and bacteroids in some ineffective nodules.     

The effect of nitrogen addition on N2-fixation and on glutamate dehydrogenase and glutamate synthase activities. in nodules and roots of soybeans inoculated with various strains of Rhizobium japonicum

The addition of nitrogen in the form of urea decreased the activitiesof glutamate dehydrogenase (GDH) and glutamate synthase (GOGAT)in root nodules of Glycine max, whereas the same addition greatlyenhanced root GDH activity. Division of nodules into a mitochondrialand bacteroid fraction indicated that the addition of nitrogenas urea, ammonia, or nitrate most greatly inhibits GDH activityin the mitochondrial fraction. Studies with plants having floralprimordia indicated that added nitrate inhibits nodular GDHmore than either ammonia or urea, while plants inoculated withan ineffective strain (non-nitrogen fixing) of Rhizobium japonicumshowed an increase in nodular GDH activity with nitrogen addition.GOGAT activity was greatly reduced after floral initiation.GDH, GOGAT, and nitrogenase activities in root nodules appearedto vary with the strain of Rhizobium japonicum used as inoculum.In general, strains which produced nodules with high GDH activityproduced bacteroids with low GOGAT activity and the strain whichproduced nodules with the lowest GDH activity produced bacteroidswith the highest GOGAT activity. (Received May 24, 1976; )     

<Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> bacteroid appendages expressed in senescing and argon-treated soybean nodules

Bradyrhizobium japonicum bacteroids in soybean nodules expressed fibrillar appendages during senescence. In both scanning and transmission electron microscopy (SEM and TEM), these structures were observed to connect adjacent bacteroids, and bacteroids to symbiotic membranes. They were 20–25 nm in diameter, 100–2,500 nm in length and were linear, branched, or part of a web-like matrix. Bacteroids expressing appendages were not uniformly distributed, but were abundant within localized regions in the senescing nodule. The root systems of nodulated greenhouse-grown plants flushed with argon induced the appendages at earlier plant ages, and they were more prolific and wide spread than those in untreated nodules. Bradyrhizobium japonicum symbiotic appendages appear to be a response to an environmental niche within senescing nodules.     

Nitrogenase activity of pea bacteroids as affected by carbohydrates and ammonium chloride

Summary Regulation and efficiency of the nitrogen-fixing system of the rhizobium-pea symbiosis were investigated. Acetylene reduction of detached root nodules was measured with various substrates added. Succinate, fumarate and malate were most effective in stimulating nitrogenase activity; glucose, pyruvate and citrate were also active. Acetylene reducing activity of detached nodules was inhibited by the addition of NH₄Cl, irrespective of the substrate present. Nitrogenase activity of isolated bacteroids was not influenced by NH₄Cl.Respiration of detached nodules was not significantly stimulated by the addition of substrates. Ammonium chloride did not influence respiration. With detached nodules and isolated bacteroids a consumption of about 16 g of carbohydrate per g of nitrogen fixed could be calculated. Detached nodules produced more hydrogen relative to the acetylene reduced than did isolated bacteroids and intact plants.Results obtained indicate that the regulation of nitrogenase activity and the efficiency of substrate consumption depend on environmental conditions.     

A block in the endocytosis of Rhizobium allows cellular differentiation in nodules but affects the expression of some peribacteroid membrane nodulins

A transposon-induced mutant (T8-1) of Bradyrhizobium japonicum (61A76) was unable to develop into the nitrogen-fixing endosymbiotic form, the bacteroid. Comparison between this mutant and T5-95, an ineffective (non-nitrogen fixing, Fix^-) mutant, confirmed that the process of bacteroid development is a distinct phase of differentiation of the endosymbiont and is independent of nitrogen fixation activity. The T8-1 mutant was able to induce normal-size nodules which differentiated two plant cell types and contained numerous infection threads. However, the infected cells were devoid of bacteroids. Electron microscopy revealed that the ends of the infection threads were broken down in a normal manner once the thread had penetrated the cells, but the mutant was not internalized by endocytosis. The lack of peribacteroid membrane (PBM) in nodules induced by this mutant was correlated with a reduced level of expression of plant genes coding for PBM nodulins. These genes were expressed in the T5-95 mutant, showing that the low expression in T8-1 was not due to the lack of nitrogen fixation. One of the PBM nodulins, nodulin-26, was found at normal levels in the nodules which lack PBM, suggesting that there are at least two developmental stages in PBM biosynthesis. These data suggest that a coordination of plant and Rhizobium gene expression is required for the release and internalization of bacteria into the PBM compartments of infected cells of nodules.author for correspondence     

The Effect of Darkness on the Leghemoglobin Content and Amino Acid Levels in the Root Nodules of Pea Plants

The dependence of the nitrogen fixing system in the root nodules of pea plants (Pisum sativum) L. cv. Torsdag II) on light induced reactions was studied. The pots of the inoculated pea plants, after the nolules had fixed nitrogen for a fornight, were transferred to a dark room. The control plants were kept under normal lighting conditions. The decay of leghemoglobin was measured after photosynthesis had ceased. In the dark the red nodules turned green in three days, when about half of the haem had been broken down. The plants in normal lighting conditions had maintained the red nodules. The appearence of leghemoglobin and bacteroids was simultaneouos. In normal lighting conditions the number of bacteroids was about 1.6 × 10⁸ per g fresh nodules. The appearance of leghemoglobin and bacteroids was simultaneous. In normal lighting conditons the number of bacteroids was bout 1.6 × 10⁸ per g fresh nodules. At the same time as the nodules turned green in the dark most of the bacteroids disappeared and the number of rod-shaped bacteria increased. After five days int the dark thenumber of bacteria of the green nodules was 2.2 × 10⁸ per g fresh nodules. A large increase of of bacteria in the nodules is one of the results after the termination of effective symbiosis. Quantitative estimations were made with an automatic amino acid analysator of the amino acid composition in the root nodules of pea plants grown in the light and of pea plants grown in the dark. Altogether 27 amino acids and amides and 3 unknown ninhydrin positive compounds were found in the free amino acid fraction. In the red N-fixing nodules asparagine, the amide of aspartic acid, was the most prominent (more than 50 per cent of the total amino acid fraction), indicating the energy charge of the nitrogen fixation. 5 days in the dark affected the proportions of the amino acids as follows. Asparagine, homoserine, γ-aminobutyric acid and ethanolamine were decreased and the most of the others increased. In the hydrolysate of the non-soluble protein fraction 25 amino acids could be detected. The proportions of the amino acids in the root nodules of light-grown and dark-grown pea plants were very similar. Hydroxyproline and α, γ-diaminopimelic acid (DAP) were found in these fraction. Most of the DAP was contained in the peptide fraction. Also hydroxyproline was found to a small extent. It was assumed that the amino acids in this fraction were derived from the peptides of both plant cells and rhizobia.     

Nitrogen-fixing symbiosis between photosynthetic bacteria and legumes

Rhizobia having photosynthetic systems form nitrogen-fixing nodules on the stem and/or root of some species of the legumes Aeschynomene and Lotononis. This review is focused on the recent knowledge about the physiology, genetics and role of the photosystem in these bacteria. Photosynthetic electron transport seems to involve reaction centers, soluble cytochrome c2 and cytochrome bc1. Anaerobically, the electron transport system becomes over-reduced. The photosynthesis genes have been partially characterized; their organization is classical but their regulation is unusual as it is activated by far-red light via a bacteriophytochrome. This original mechanism of regulation seems well adapted to promote photosynthesis during stem symbiosis. Photosynthesis plays a major role in the efficiency of stem nodulation. It is also observed that infrared light stimulates nitrogen fixation in nodules containing photosynthetic bacteroids, suggesting that photosynthesis may additionally provides energy for nitrogen fixation, allowing for more efficient plant growth. Other aspects of these bacteria are discussed, in particular their taxonomic position and nodulation ability, the role of carotenoids and the potential for application of photosynthetic rhizobia in rice culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Actual and potential nitrogen fixation ofAlbizia rhizobium symbioses as affected by nitrate

Actual nitrogen fixation of root nodules of differentAlbizia-rhizobium symbioses, was compared with the potential nitrogen fixation of isolated bacteroids. The potential nitrogen fixation exceeded actual nitrogen fixation in all symbionts. After addition of nitrate the actual nitrogen fixation decreased more than did potential nitrogen fixation in effective symbiosis, whereas in a less effective symbiosis, the actual and potential nitrogen fixation increased as a result of better photosynthate supply to the roots and nodules. As confirmed by correlation analysis, the nitrogen fixation and photosynthetic yield of suboptimum symbioses were relatively enhanced by dressing with inorganic nitrogen fertilizer.     

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.     

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.     

Symbiotic properties of C4-dicarboxylic acid transport mutants of Rhizobium leguminosarum.

The transport of succinate was studied in bacteroids of an effective, streptomycin-resistant strain (GF160) of Rhizobium leguminosarum. High levels of succinate transport occurred, and the kinetics, specificity, and sensitivity to metabolic inhibitors were similar to those previously described for free-living cells. The symbiotic properties of two transposon (Tn5)-mediated C4-dicarboxylate transport mutants (strains GF31 and GF252) were determined. Strain GF31 formed ineffective nodules, and bacteroids from these nodules showed no succinate transport activity. Strain GF252 formed partially effective nodules, and bacteroids from these nodules showed about 50% of the succinate transport activity of the parent bacteroids. Another dicarboxylic acid transport mutant (Dct-), strain GFS5, isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, formed ineffective nodules. The ability to form ineffective nodules in strains GF31 and GFS5 was shown to correlate with the Dct- phenotype. The data indicate that the presence of a functional C4-dicarboxylic acid transport system is essential for N2 fixation to occur in pea nodules.     

The Formation of Nitrogen-Fixing Bacteroids Is Delayed but Not Abolished in Soybean Infected by an [alpha]-Ketoglutarate Dehydrogenase-Deficient Mutant of Bradyrhizobium japonicum

A mutant strain of Bradyrhizobium japonicum USDA 110 devoid of [alpha]-ketoglutarate dehydrogenase activity (LSG184) was used to test whether this tricarboxylic acid cycle enzyme is necessary to support nitrogen fixation during symbiosis with soybean (Glycine max). LSG184 formed nodules about 5 d later than the wild-type strain, and the nodules, although otherwise normal in structure, contained many fewer infected host cells than is typical. At 19 d after inoculation cells infected with the mutant strain were only partially filled with bacteroids and showed large accumulations of starch, but by 32 d after inoculation the host cells infected with the mutant appeared normal. The onset of nitrogen fixation was delayed about 15 d for plants inoculated with LSG184, and the rate, on a per nodule fresh weight basis, reached only about 20% of normal. However, because nodules formed by LSG184 contained only about 20% of the normal number of bacteroids, it could be inferred that the mutant, on an individual bacteroid basis, was fixing nitrogen at near wild-type rates. Therefore, the loss of [alpha]-ketoglutarate dehydrogenase in B. japonicum does not prevent the formation or the functioning of nitrogen-fixing bacteroids in soybean.     

Characterization of the anomalous infection and nodulation of subterranean clover roots by Rhizobium leguminosarum 1020

Anomalous nodulation of Trifolium subterraneum (subterranean clover) roots by Rhizobium leguminosarum 1020 was examined as a model of modified host-specificity in a Rhizobium-legume symbiosis. Consistent with previous reports, these nodules (i) appeared most often at sites of secondary root emergence, (ii) were ineffective in nitrogen fixation and (iii) were as numerous as nodules formed by an effective Rhizobium trifolii strain. R. leguminosarum 1020, grown on agar plates or in the clover root environment, did not bind the white clover lectin, trifoliin A. This strain did not attach in high numbers, and did not induce shepherd's crooks or infection threads, in subterranean clover root hairs. However, R. leguminosarum 1020 did cause branching, moderate curling and other deformations of root hairs. The bacteria probably entered the clover root through breaks in the epidermis at sites of lateral root emergence. The anomalous nodulation was inhibited by nitrate. Only trace amounts of leghaemoglobin were detected in the nodules by Western blot analysis. The nodules were of the meristematic type and initially contained well-developed infection, bacteroid and senescent zones. Infection threads were readily found in the infection zone of the nodule. However, the bacteroid-containing tissue senesced more rapidly than in the effective symbiosis between subterranean clover and R. trifolii 0403. This anomalous nodulation of subterranean clover by R. leguminosarum 1020 suggests a naturally-occurring alternative route of infection that allows Rhizobium to enlarge its host range.     

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.     

Development of nodules of Glycine max infected with an ineffective strain of Rhizobium japonicum

Bacteroids in ineffective (nitrogenase negative) nodules of Glycine max, infected with Rhizobium japonicum 61-A-24, as compared to those in effective nodules are characterized by reduced specific activities of alanine dehydrogenase to 15%, of 3-hydroxybutyrate dehydrogenase to 50%, and an increase of glutamine synthetase to 400%. In the plant cytoplasm of ineffective nodules, glutamine synthetase activity is reduced to 10–30%, glutamate dehydrogenase to 50–70%, and the aspartate aminotransferase and alanine aminotransferase are enhanced to 120–200%, depending on the age of the nodules. The total pool of soluble amino acids is reduced to 52 mol per g nodule fresh weight, as compared to 186 mol in effective nodules, with a replacement of asparagine (42 mol% of the amino acids) by an unknown amino compound. This compound is absent in nitrogenase, repressed and derepressed, free-living Rhizobium japonicum cells and in the uninfected root tissue. In nitrogenase derepressed, as compared to the repressed free-living cells of Rhizobium japonicum 61-A-101, arginine shows the most obvious change with a reduction to less than one tenth. The ultrastructure of the ineffective nodule is different from the effective organ even in the early stages. The membrane envelopes of the infection vacuoles are decomposing in heavily infected cells within 18 to 20 d after infection. In lightly infected cells very large vacuoles develop with only a few bacteroids inside. No close associations of cristae-rich mitochondria with amyloplasts are observed as in effective nodules. The uninfected cells keep their large starch granules even 40 d after infection. Some poly--hydroxybutyrate accumulation in the bacteroids is observed but only in the early stages, and it is almost absent in old nodules (40 d). At this age the infected cells are obviously compressed by uninfected cells, whereas in effective nodules with nitrogenase activity and leghaemoglobin formation, the infected cells have a much higher osmotic pressure than the neighbouring uninfected cells.Abbreviations PHBA poly--hydroxybutyric acid Prof. Dr. A. Pirson on the occasion of his 70th birthday