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

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

Effect of nutrient medium pH on symbiotic nitrogen fixation byRhizobium leguminosarum andPisum sativum

Summary Experiments were performed to measure the pH-sensitive steps in nodulation and symbiotic fixation byPisum sativum and isolate RP-212-1 ofRhizobium leguminosarum. An aeroponic system with rigorous pH control was used to obtain numerous effective nodules. After exposure to various pH levels, the following responses were measured: (1) legume root growth and development, (2) survival and growth rate of a single effective bacterial isolate, (3) degree of nodulation, (4) rate of nitrogen fixation, (5) plant biomass, and (6) nitrogen content of plants. Both bacterial growth and root development were adequate at all pH levels from 4.4 to 6.6, but efficient nodulation and nitrogen fixation did not occur at pH 4.8 and below. The processes required for symbiosis were about 10 times as sensitive to acidity as either bacterial growth or root growth alone. Nodulation was the most acid-sensitive step.     

Nitrate effects on the nodulation of legumes inoculated with nitrate-reductase-deficient mutants of Rhizobium

The effect of nitrate on the symbiotic properties of nitrate-reductase-deficient mutants of a strain of cowpea rhizobia (32H1), and of a strain of Rhizobium trifolii (TA1), were examined; the host species were Macroptilium atropurpureum (DC.) Urb. and Trifolium subterraneum L. Nitrate retarded initial nodulation by the mutant strains to an extent similar to that found with the parent strains. It is therefore unlikely that nitrite produced from nitrate by the rhizobia, plays a significant role in the inhibition of nodulation by nitrate. Nitrite is an inhibitor of nitrogenase, and its possible production in the nodule tissue by the action of nitrate reductase could be responsible for the observed inhibition of nitrogen fixation when nodulated plants are exposed to nitrate. However, the results of this investigation show that nitrogen fixation by the plants nodulated by parent or mutant strains was depressed by similar amounts in the presence of nitrate. No nitrite was detected in the nodules. Nodule growth, and to a lesser extent, the nitrogenase specific activity of the nodules (mol C₂H₄g^–1 nodule fr. wt. h^–1), were both affected by the added nitrate.     

Nitrate levels affect the development of the black locust-Rhizobium symbiosis

Summary Experiments with black locust (Robinia pseudoacacia L.) seedlings grown under strictly controlled laboratory conditions indicated that the availability of nitrate has a marked impact on nitrogen fixation. When nitrate concentrations were very low, both nodulation and seedling growth were impaired, whereas nitrate concentrations high enough to promote plant growth strongly inhibited symbiotic nitrogen fixation. When nitrate was added to the growth medium after infection, nodulation and nitrogen fixation of the seedlings decreased. This effect was even more marked when nitrate was applied before infection with rhizobia. Higher nitrogen concentrations also reduced nodule number and nodule mass when applied simultaneously with the infecting bacteria. The contribution of symbiotic nitrogen fixation to black locust shoot mass by far exceeded its effects on shoot length and root mass. When nitrate availability was very low, specific nitrogen fixation (i. e. nitrogenase activity per nodule wet weight) was improved with increasing nitrogen supply, but rapidly decreased with higher nitrogen concentrations.     

Cycling of amino compounds in symbiotic lupin

The composition of amino acids was determined in the xylem andphloem sap of symbiotic lupins grown under a variety of treatmentsdesigned to alter the rate of nitrogen fixation. Asparaginewas the major amino acid in both xylem and phloem with glutamine,glutamate and aspartate also major components. GABA had a highconcentration in the xylem while valine was a major componentin the phloem. Exposure to combined nitrogen in the form ofeither ammonium or nitrate caused a reduction in specific nitrogenaseactivity and was associated with subsequent changes in bothof the translocated saps. Inhibiting nitrogen fixation by exposingnodules to oxygen produced a lower amide to amine ratio in thexylem sap (1.3:1) compared with control and nitrate ratios (2.6:1)and ammonium ratios (7.1:1). Similar ratios for amide aminewere also observed in the phloem sap. Labelling studies using¹⁵N₂ to follow nitrogen fixation, ammonium assimilation andamino acid transport have shown rapid accumulation of labelinto glutamine with subsequent enrichment in glutamate, aspartate,alanine, and GABA. Asparagine was found in high concentrationsin nodules and became slowly enriched. Labelled nitrogen fixedand assimilated in nodules was detected 40 min later in stemxylem extracts, largely as the amides glutamine and asparagine.These experiments provide evidence that large amounts of nitrogenouscompounds are cycled through the root nodules of symbiotic plants(contributing approximately 50% of xylem N) and that differencesin the composition of the phloem sap may influence nodule growthand activity. Key words: Nitrogen fixation, nitrogen translocation, isotope labelling, legumes, GC-MS     

Sensitivity of the common bean (Phaseolus vulgaris L.) symbiosis to high soil temperature

Summary Experiments were done to test whether N fixation is more sensitive to high soil temperatures in common bean than in cowpea or soybean. Greenhouse experiments compared nodulation, nitrogenase activity, growth and nitrogen accumulation of several host/strain combinations of common bean with the other grain legumes and with N-fertilization, at various root temperatures. Field experiments compared relative N-accumulation (in symbiotic relative to N-fertilized plants) of common bean with cowpea under different soil thermal regimes. N-fertilized beans were unaffected by the higher temperatures, but nitrogen accumulation by symbiotic beans was always more sensitive to high root temperatures (33°C, 33/28°C, 34/28°C compared with 28°C) than were cowpea and soybean symbiosis. Healthy bean nodules that had developed at low temperatures functioned normally in acetylene reduction tests done at 35°C. High temperatures caused little or no suppression of nodule number. However, bean nodules produced at high temperatures were small and had low specific activity. ForP. vulgaris some tolerance to high temperature was observed among rhizobium strains (e.g., CIAT 899 was tolerant) but not among host cultivars. Heat tolerance ofP. acutifolius andP. lunatus symbioses was similar to that of cowpea and soybean. In the field, high surface soil temperatures did not reduce N accumulation in symbiotic beans more than in cowpea, probably because of compensatory nodulation in the deeper and cooler parts of the soil.     

Nitrogen control of bacterial signal production in Rhizobium meliloti-alfalfa symbiosis

Under nitrogen-depleted conditions nitrogen-fixing soil bacteria of the family Rhizobiaceae are able to induce symbiotic nodules on the roots of leguminous plants where bacteroids convert atmospheric nitrogen to ammonia. The presence of exogenous nitrogen source inhibits the development and the functioning of bacterium-plant symbiosis. Earlier experiments demonstrated that nitrate inhibited all stages of symbiotic interaction, affecting primarily the host functions. The investigation of the possible involvement of the microsymbiont in nitrogen regulation showed that two signalling steps were controlled by ammonium. The synthesis of the first bacterial signal, the Nod factor was repressed by ammonium. The nitrogen signal is conveyed to nodulation (nod) genes by the general nitrogen regulatory (ntr) system and by the nodD3-syrM self-amplifying system. The fine control also involves a negative regulatory factor, ntrR. When ntrR is mutated, more efficient nodule formation and nitrogen fixation is observed in symbiosis with alfalfa even in the presence of ammonium. The biosynthesis of the second bacterial signal succinoglycan is also controlled by ammonium. SyrM, a common regulatory factor for nod and exo gene expression, may contribute to the adjustment of the amount of succinoglycan and the ratio of its biologically active form.     

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.     

MALDI mass spectrometry‐assisted molecular imaging of metabolites during nitrogen fixation in the Medicago truncatula–Sinorhizobium meliloti symbiosis

Symbiotic associations between leguminous plants and nitrogen‐fixing rhizobia culminate in the formation of specialized organs called root nodules, in which the rhizobia fix atmospheric nitrogen and transfer it to the plant. Efficient biological nitrogen fixation depends on metabolites produced by and exchanged between both partners. The Medicago truncatula–Sinorhizobium meliloti association is an excellent model for dissecting this nitrogen‐fixing symbiosis because of the availability of genetic information for both symbiotic partners. Here, we employed a powerful imaging technique – matrix‐assisted laser desorption/ionization (MALDI)/mass spectrometric imaging (MSI) – to study metabolite distribution in roots and root nodules of M. truncatula during nitrogen fixation. The combination of an efficient, novel MALDI matrix [1,8–bis(dimethyl‐amino) naphthalene, DMAN] with a conventional matrix 2,5–dihydroxybenzoic acid (DHB) allowed detection of a large array of organic acids, amino acids, sugars, lipids, flavonoids and their conjugates with improved coverage. Ion density maps of representative metabolites are presented and correlated with the nitrogen fixation process. We demonstrate differences in metabolite distribution between roots and nodules, and also between fixing and non‐fixing nodules produced by plant and bacterial mutants. Our study highlights the benefits of using MSI for detecting differences in metabolite distributions in plant biology.     

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.     

Nitric oxide in legume-rhizobium symbiosis

Nitric oxide (NO) is a gaseous signaling molecule with a broad spectrum of regulatory functions in plant growth and development. NO has been found to be involved in various pathogenic or symbiotic plant-microbe interactions. During the last decade, increasing evidence of the occurrence of NO during legume-rhizobium symbioses has been reported, from early steps of plant-bacteria interaction, to the nitrogen-fixing step in mature nodules. This review focuses on recent advances on NO production and function in nitrogen-fixing symbiosis. First, the potential plant and bacterial sources of NO, including NO synthase-like, nitrate reductase or electron transfer chains of both partners, are presented. Then responses of plant and bacterial cells to the presence of NO are presented in the context of the N₂-fixing symbiosis. Finally, the roles of NO as either a regulatory signal of development, or a toxic compound with inhibitory effects on nitrogen fixation, or an intermediate involved in energy metabolism, during symbiosis establishment and nodule functioning are discussed.     

Effects of disease resistance genes on Rhizobium symbiosis in an annual legume

Summary The evolution of disease resistance in plants may be constrained if genes conferring resistance to pathogens interfere with plant responses toward other, nonpathogenic organisms. To test for such effects, we compared symbiotic nitrogen fixation in Amphicarpaea bracteata plants that differed at a major locus controlling resistance to the pathogen Synchytrium decipiens. Both resistant and susceptible plant genotypes nodulated successfully and grew significantly better in the presence of Rhizobium, although growth enhancement by Rhizobium was altered by different levels of nitrate fertilization. Plants homozygous for disease resistance achieved 2% higher growth than susceptible homozygotes across all treatments, but this difference was not significant. Resistant and susceptible plant genotypes did not differ in the mean number of nodules formed per plant or in nodule diameter. However, there was highly significant variation among replicate families within each disease resistance category for both nodulation characteristics. These results imply that genetic variation exists among A. bracteata plants both for diease resistance and for traits affecting symbiotic nitrogen fixation. However, there were no evident pleiotropic effects of disease resistance genes on the plant-Rhizobium symbiosis.     

Recovery of nodulated cowpea plants (Vigna unguiculata (L.) Walp.) from waterlogging during vegetative growth

Summary Waterlogging pot-grown cowpea plants for eight days (24–32 from sowing) during the period of rapid vegetative growth when symbiotic nitrogen fixation rates are also increasing at maximum rate had effects on anatomical, morphological and physiological attributes of the legume system viz reduced nodule production and increased nodule cortication, stimulated adventitious root formation at, or near, the water table but killed older submerged roots and nodules, caused the main stem to become hypertrophic just above the water line, accelerated senescence of the lowermost leaves and delayed branch formation. After re-draining the rooting medium nodule growth and symbiotic fixation activity occurred at the expense of rapid recovery by the host plant. The physiological basis for these findings in relation to identifying cowpea cultivars more tolerant of waterlogged soils is discussed.One of a series of papers describing work undertaken in a collaborative project with the International Institute of Tropical Agriculture, Nigeria, sponsored by the U.K. Ministry of Overseas Development.     

Symbiotic Nitrogen Fixation and Ammonium Nitrogen Assimilation in rrrbrb-, rrRbRb-, and RRrbrb-Pea Mutants

Wrinkled-seeded pea mutants (Pisum sativum L., genotypes rrrbrb-, rrRbRb-, and RRrbrb-) have seeds with reduced, but different, starch content and modified starch properties. Analysis of these mutants revealed an enhanced capacity of root nodules for symbiotic nitrogen fixation and of host plant organs for assimilation of ammonium nitrogen. This observation was confirmed by morphological data on organization of symbiotic system, by elevated nitrogenase activity, high protein accumulation in plants due to nitrogen fixation, and by enhanced activity of glutamine synthase in leaves and glutamate dehydrogenase in roots of mutants, as compared with the organs of wild-type pea. It is supposed that the aforementioned advantages of mutants are related to accumulation in seeds of elevated protein reserves that satisfy their demand for nitrogen during formation of symbiotic systems.     

Bacterial RuBisCO is required for efficient Bradyrhizobium/Aeschynomene symbiosis

Rhizobia and legume plants establish symbiotic associations resulting in the formation of organs specialized in nitrogen fixation. In such organs, termed nodules, bacteria differentiate into bacteroids which convert atmospheric nitrogen and supply the plant with organic nitrogen. As a counterpart, bacteroids receive carbon substrates from the plant. This rather simple model of metabolite exchange underlies symbiosis but does not describe the complexity of bacteroids' central metabolism. A previous study using the tropical symbiotic model Aeschynomene indica/photosynthetic Bradyrhizobium sp. ORS278 suggested a role of the bacterial Calvin cycle during the symbiotic process. Herein we investigated the role of two RuBisCO gene clusters of Bradyrhizobium sp. ORS278 during symbiosis. Using gene reporter fusion strains, we showed that cbbL1 but not the paralogous cbbL2 is expressed during symbiosis. Congruently, CbbL1 was detected in bacteroids by proteome analysis. The importance of CbbL1 for symbiotic nitrogen fixation was proven by a reverse genetic approach. Interestingly, despite its symbiotic nitrogen fixation defect, the cbbL1 mutant was not affected in nitrogen fixation activity under free living state. This study demonstrates a critical role for bacterial RuBisCO during a rhizobia/legume symbiotic interaction.     

Two effects of combined nitrogen on the adhesion of Rhizobium etli to bean roots

Combined forms of nitrogen negatively influence rhizobia-legume symbiosis. The effects of combined nitrogen are known for nodulation and dinitrogen (N₂) fixation, but little is known about the effect on preinfection events. Here, we studied the effects of combined nitrogen on the adhesion of Rhizobium etli to common bean (Phaseolus vulgaris L.) roots. When potassium nitrate (KNO₃) or sodium glutamate was added to an incubation mixture of rhizobia and plants that were previously grown in nitrogen-free solution, rhizobial adhesion to roots was stimulated. However, the rhizobial adhesion to bean roots that were previously grown with 10 mM KNO₃ was reduced by half. A fraction of the bean root exudates, which is thermolabile and has molecular mass larger than 12 kDa stimulated rhizobial adhesion, but this stimulatory activity was lost in root exudates obtained with 10 mM KNO₃. Thus, the inhibition of symbiosis in response to combined nitrogen may be controlled by the plant at the preinfection stage as well.     

Loss‐of‐function of ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (APN1) in Lotus japonicus restricts efficient nitrogen‐fixing symbiosis with specific Mesorhizobium loti strains

The nitrogen‐fixing symbiosis of legumes and Rhizobium bacteria is established by complex interactions between the two symbiotic partners. Legume Fix^– mutants form apparently normal nodules with endosymbiotic rhizobia but fail to induce rhizobial nitrogen fixation. These mutants are useful for identifying the legume genes involved in the interactions essential for symbiotic nitrogen fixation. We describe here a Fix^– mutant of Lotus japonicus, apn1, which showed a very specific symbiotic phenotype. It formed ineffective nodules when inoculated with the Mesorhizobium loti strain TONO. In these nodules, infected cells disintegrated and successively became necrotic, indicating premature senescence typical of Fix^– mutants. However, it formed effective nodules when inoculated with the M. loti strain MAFF303099. Among nine different M. loti strains tested, four formed ineffective nodules and five formed effective nodules on apn1 roots. The identified causal gene, ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (LjAPN1), encodes a nepenthesin‐type aspartic peptidase. The well characterized Arabidopsis aspartic peptidase CDR1 could complement the strain‐specific Fix^– phenotype of apn1. LjAPN1 is a typical late nodulin; its gene expression was exclusively induced during nodule development. LjAPN1 was most abundantly expressed in the infected cells in the nodules. Our findings indicate that LjAPN1 is required for the development and persistence of functional (nitrogen‐fixing) symbiosis in a rhizobial strain‐dependent manner, and thus determines compatibility between M. loti and L. japonicus at the level of nitrogen fixation.     

Development of the nitrogen fixing apparatus in the legumes,Centrosema pubescens Benth., and Vigna unguiculata L. Walp.

The sequence of events leading up to the establishment of symbiotic nitrogen-fixation were studied in two tropical legumes, Centrosema pubescens Benth, and Vigna unguiculata L. Walp. Parameters measured included fresh and dry weights, chlorophyll and leghaemoglobin contents, as well as the activities of NADH-nitrate reductase (EC 1.6.6.1), and nitrogenase (nitric-oxide reductase-EC 1.7.99.2) in plants that were inoculated with suitable rhizobia or which were watered with potassium nitrate. Dry weight and photosynthetic activity of both species followed the sigmoidal pattern which is characteristic of most plants. Growth was little different in either a qualitative or quantitative sense whether nitrogen was supplied as nitrate or through dinitrogen fixation. Although the biochemical sequence of events was dependent on the limiting sensitivities of the individual assays used, the data suggest that nitrate reductase is the first measurable enzymatic activity in the nodules (and roots), followed by acetylene reduction and leghaemoglobin in that order. It is possible therefore, that low levels of symbiotic nitrogen fixation occur in the nodules in the absence of leghaemoglobin. Nitrate reductase activity in C. pubescens nodules was negatively exponentially correlated with nitrogenase activity of the same nodules, suggesting a changing metabolism in old nodules. These data are discussed in terms of environmental and physical factors known to control nitrogen fixation.     

Influence of ammonium and nitrate nitrogen on nitrogenase activity of pea plants as affected by light intensity and sugar addition

Summary Addition of ammonium chloride or potassium nitrate to nodulated pea plants resulted in a decrease in acetylene-reducing activity. Both nodule growth and specific activity of the nodules were diminished. Acetylene-reducing activity of isolated bacteroids, treated with EDTA-toluene and supplied with ATP and dithionite, had not decreased after a 3-day treatment of the plants with NH₄Cl or KNO₃. The effect of combined nitrogen could be counteracted by raising the light intensity or by the addition of sucrose to the growth medium. The latter treatment reduced the nitrogen uptake by the plants. It is concluded that combined nitrogen affects symbiotic nitrogen fixation via the carbohydrate supply to the bacteroids.