Nodule-specific host proteins in effective and ineffective root nodules of Pisum sativum

Nodule-specific root proteins – so called nodulins – were identified in root nodules of pea plants by an immunological assay. Nodulin patterns were examined at different stages of nodule development. About 30 nodulins were detectable during development. Some were preferentially synthesized before nitrogen fixation started, whereas the majority were synthesized concomitantly with leghaemoglobin. Some of the nodulins were located within the peribacteroid membrane. Ineffective Rhizobium strains (a natural nod⁺fix^- and a pop ^-fix^-) appeared to be useful in studying the expression of nodulin genes. Synthesis of some nodulins was repressed in ineffective root nodules, indicating that nodulins are essential for the establishment of nitrogen fixation. In both types of ineffective root nodules, leghaemoglobin synthesis was not completely repressed. Low amounts of leghaemoglobin were always detected in young ineffective root nodules whereas in old nodules no leghaemoglobin was present.     

Growth and root nodule nitrogenase activity of Pisum sativum as influenced by transpiration

The influence of shoot transpiration on the rates of growth and nitrogen fixation was investigated in Pisum sativum L. cv. Rondo. In short term experiments, rates of transpiration and acetylene reduction of intact plants were measured simultaneously, using air-tight perspex vessels enclosing the basal part of the nodulated root. In long term experiments, accumulation of dry matter and reduced nitrogen in the plant were determined as well. Transpiration rate changed diurnally and was varied by manipulating the vapour saturation deficit or the flow rate of the air in the growth cabinet. The rate of acetylene reduction declined after subjecting intact plants to high transpiration rates. This decline was accompanied by a desiccation of the root nodules. Dry matter and reduced nitrogen accumulation were not affected by transpiration rate. At low transpiration rate reduced nitrogen content of the root nodules was higher than at high transpiration rate. However, in these nodules the rate of acetylene reduction was not significantly affected. It is concluded that the nitrogenase activity of pea root nodules is insensitive to changes in the flow rate and the organic N concentration of the xylem sap within a wide range of transpiration conditions under the applied growth conditions.     

Reduction of ferric leghemoglobin in soybean root nodules

Callus tissue cultures were developed from apical meristem regions of tumor-like ineffective root nodules of alfalfa. Callus growth was a function of tissue source and hormone composition and concentration. Callus derived from ineffective nodules also were shown not to contain Rhizobium meliloti.

Glutamate dehydrogenase, glutamine synthetase, glutamate synthase, glutamate oxaloacetate transaminase and phosphoenolpyruvate carboxylase activities were present in callus cultures and in the respective nodule source used for callus induction. The mean specific activity of all enzymes evaluated was higher in callus cultures than in ineffective nodules. Quantitative but not qualitative differences in enzyme activities were evident between ineffective nodules and callus derived from these nodules. Tissue cultures derived from ineffective nodules may provide a model system to evaluate host plant-Rhizobium interactions.

     

Ferric leghemoglobin reductase from soybean root nodules

An NADH: (acceptor) oxidoreductase from the cytosol of soybean root nodules was purified by ammonium sulfate fractionation, hydroxylapatite adsorption, and Sephacryl S-200 Superfine chromatography. The native molecular weight of the reductase was found to be 100,000 by analytical gel filtration and 83,000 by equilibrium ultracentrifugation. The subunit molecular weight was 54,000 as determined by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The pI of the enzyme was 5.5. With ferric leghemoglobin (Lb) as the substrate, nearly identical initial velocities were obtained using either CO or O2 to ligate the enzymatically produced ferrous leghemoglobin. With CO as the ligand in the reaction, the product of the enzyme-catalyzed, NADH-dependent reduction of ferric Lb was spectrally identified as LbCO. Initial velocity was a linear function of increasing enzyme concentration. NADPH was only 31% as effective an electron donor as NADH as determined by initial velocity. The Michaelis constants (Km) for ferric Lba and NADH were 9.5 and 18.8 microM, respectively. Myoglobin, Lba, Lbc1, Lbc2, Lbc3, and Lbd were reduced at similar rates by the reductase. At pH 5.2, acetate-bound ferric Lb and nicotinate-bound ferric Lb were reduced by the enzyme at 83 and 5%, respectively, of rates observed in the absence of these ligands. The rate of enzymatic reduction of ferric Lb was constant between pH 6.5 and 7.6 but increased approximately threefold at pH 5.2. The results indicate that the NADH: (acceptor) oxidoreductase could be identified as a ferric Lb reductase.     

Initial proliferation of cortical cells in the formation of root nodules in Pisum sativum L.

Summary Root nodule initiation in Pisum sativum begins with cell divisions in the inner cortex at some distance from the advancing infection thread. After penetrating almost the entire cortex, the branches of the thread infiltrate the meristematic area previously initiated in the inner cortical cells. These cells are soon invaded by bacteria released from the infection thread and subsequently differentiate into non-dividing, bacteriod-containing cells. As the initial meristematic centre in the inner cortex is thus lost to bacteroid formation, new meristematic activity is initiated in neighbouring cortical cells. As development proceeds, more cortical layers contribute to the nodule, with the peripheral layer and apical meristem of the nodule not invaded by bacteria.Lateral root primordia are initiated in a region separate from that in which nodules are formed, with the lateral primordia being closer to the root apex. This is interpreted to indicate that the physiological basis for nodule initiation is distinct from that for initiation of lateral roots. The role of a single tetraploid cell in nodule initiation is refuted, as is the existence of incipient meristematic foci in the root. It is suggested that the tetraploid cells in nodule meristems arise from pre-existing endoreduplicated cells, or by the induction of endoreduplication in diploid cortical cells by Rhizobium.     

Fluctuations of nitrogenase and cytosol glutamine synthetase activity in pea (Pisum sativum L.) nodules in the course of vegetation

Changes in glutamine synthetase activity located in the cytosol of root nodules were followed in pea (Pisum sativum L.) plants in relation to their nitrogenase activity. The highest glutamine synthetase activity was found in young nodules (15 days after inoculation) and its changes in 17-to 45-day-old plants showed a positive correlation with nitrogenase activity. In contrast to nitrogenase activity, changes in glutamine synthetase activity during the day and night period could not be unequivocally interpreted in terms of diurnal fluctuation.     

Oxidation and reduction of leghemoglobin in root nodules of leguminous plants

Reactions involving changes that affect the function of leghemoglobin (Lb) are reviewed. The chemical nature of Lb and conditions inside nodules, such as slightly acid pH and the presence of metal ions, chelators, and toxic metabolites (nitrite, superoxide radical, peroxides), are conducive for oxidation of ferrous Lb (Lb²⁺) or its oxygenated form (LbO₂) to nonfunctional ferric Lb (Lb³⁺) and ferryl Lb. Because Lb³⁺ is nearly nonexistent in nodules and undergoes observable reduction in vivo, mechanisms must operate in nodules to maintain Lb in the Lb²⁺ state. Redox reactions of Lb are mediated, for the most part, by activated oxygen species: (a) oxidation of LbO₂ to Lb³⁺ involves superoxide; (b) excess peroxide oxidizes LbO₂ and Lb³⁺ to ferryl Lb and may cause breakdown of heme, release of iron, and generation of hydroxyl radicals (protein radicals may be formed in this process); (c) enzymatic reduction of Lb³⁺ requires active flavin and thiol groups and involves formation of peroxide; and (d) direct reduction of Lb³⁺ by NADH is mediated by superoxide and peroxide. Transition metal ions and certain small molecules of nodules such as flavins may act as intermediate electron carriers between NADH and Lb³⁺, increasing the rate of reaction, which then proceeds via superoxide or flavin radicals, respectively.     

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.     

Abscisic acid inhibition of root nodule initiation in Pisum sativum

Summary The effect of exogenous abscisic acid (ABA) on root nodule formation in Pisum sativum cv. Alaska was examined. ABA supplied to the roots at 1.9×10^-6M reduced the number of nodules/plant 61% without affecting root or shoot growth. The first noticeable inhibition of nodulation occurred at 3.8×10^-7M ABA. ABA at a concentration of 1.9×10^-6M inhibited neither root hair formation nor infection of root hairs by Rhizobium leguminosarum. Similar numbers of infection threads penetrated the cortex in both control and treated plants. ABA concentrations of 3.8×10^-6M had no effect on the doubling time or maximum density of R. leguminosarum in pure cultures. Normal nodule formation involves a polyploid cortical proliferation. This response to rhizobial infection can be imitated by culturing 1-mm pea-root segments on a medium containing 4.7×10^-6M kinetin. Under these conditions a highly significant reduction in the number of polyploid mitoses after 72 h is produced by 3.8×10^-8M ABA. A maximum inhibition of 68% was found with 3.8×10^-6M ABA. A similar range of ABA concentrations also inhibited the cytokinin-induced cell division in soybean callus. It is concluded that ABA reduces the number of root nodules/plant by inhibiting the cortical cell divisions required for root nodule formation.     

Nicotinate, nicotinamide, and the reactivity of leghemoglobin in soybean root nodules

Nicotinate has been postulated to interfere with the binding of O₂ to ferrous leghemoglobin in soybean (Glycine max) root nodules. For such a function, the levels of nicotinate in nodules must be sufficiently high to bind a significant amount of leghemoglobin. We have measured levels of nicotinate, nicotinamide, and leghemoglobin in soybean nodules from plants 34 to 73 days after planting in a glasshouse. On a per gram nodule fresh weight basis, levels between 10.4 and 21 nanomoles for nicotinate, 19.2 and 37.8 nanomoles for nicotinamide, and 170 to 280 nanomoles for leghemoglobin were measured. Even if all the nicotinate were bound to ferrous leghemoglobin, only 11% or less of the total leghemoglobin would be unavailable for binding O₂. Using the measured levels of nicotinate and a pH of 6.8 in the cytosol of presenescent soybean nodules, we estimate that the proportion of ferrous leghemoglobin bound to nicotinate in such nodules would be less than 1%. These levels of nicotinate are too low to interfere with the reaction between ferrous leghemoglobin and O₂ in soybean root nodules.     

Capacity for fermentation in roots and Rhizobium nodules of Pisum sativum L.

The aim of this work was to compare the capacities for fermentation and synthesis of malate from phosphoenolpyruvate in roots and Rhizobium nodules of Pisum sativum. The nodules and the cortices and apices of roots had similar activities of glycolytic enzymes and enzymes of ethanolic and lactic fermentation when expressed on a protein basis. The activity of phosphoenolpyruvate carboxylase was similar in nodules and apices, and three to four fold lower in cortices. All three tissues had very high activities of malate dehydrogenase, significant activity of NADP-malic enzyme, and no detectable activity of phosphoenolpyruvate carboxykinase. These results do not support the belief that nodules have a substantially greater capacity to convert phosphoenolpyruvate to malate than roots, or that there are major qualitative differences in the pathways of fermentation of nodules and roots.Abbreviation PEP phosphoenolpyruvate     

Variation in nitrogenase and hydrogenase activity of alaska pea root nodules

Hydrogenase activity of root nodules in the symbiotic association between Pisum sativum L. and Rhizobium leguminosarum was determined by incubating unexcised nodules with tritiated H₂ and measuring tissue HTO. Hydrogenase activity saturated at 0.50 millimolar H₂ and was not inhibited by the presence of 0.10 atmosphere C₂H₂, which prevented H₂ evolution from nitrogenase. Total H₂ production from nitogenase was estimated as net H₂ evolution in air plus H₂ exchange in 0.10 atmosphere C₂H₂. Although such an estimate of nitrogenase function may not be quantitatively exact, due to uncertain relationships between H₂ exchange and H₂ uptake activity of hydrogenase, differences observed in H₂ exchange under various conditions represent an indication of changes in hydrogenase activity. Hydrogenase activity was lower in associations grown under higher photosynthetic photon flux densities and decreased relative to total H₂ production by nitrogenase. Total H₂ production and hydrogenase activity were maximum 28 days after planting. Thereafter, hydrogenase activity and H₂ production declined, but the potential proportion of nitrogenase-produced H₂ recovered by the uptake hydrogenase system increased. Of five R. leguminosarum strains tested two possessed hydrogenase activity. Strains which had the potential to reassimilate H₂ had significantly higher rates of N₂ reduction than those which did not exhibit hydrogenase activity.     

Membrane lipid peroxidation, nitrogen fixation and leghemoglobin content in soybean root nodules

Membrane lipids in soybean nodules may undergo oxidative degradation resulting in the loss of membrane structural integrity and physiological activities. One of the final products of lipid peroxidation is malondialdehyde (MDA), which can react with thiobarbituric acid (TBA) in vitro to form a chromogenic adduct, a Schiff base product that can be measured spectrophotometrically. MDA formation was quantified in the nodules as well as in the adjacent root tissue. Lipid peroxidation was initially high in soybean nodules induced by Bradyrhizobium japonicum, but sharply declined following an increase in both leghemoglobin content and nitrogen fixation rate. Lipid peroxidation was 2 to 4 times higher in the nodules than in their corresponding adjoining root tissue. Malondialdehyde levels in ineffective nodules were 1.5 times higher than those in effective nodules. MDA formation was also shown to occur in the ‘leghemoglobin-free’ cytosolic fraction, the ‘leghemoglobin’ fraction, and the nodule tissue pellet. Antioxidants, such as reduced ascorbic acid, glutathione, and 8-hydroxyquinoline, caused a partial suppression of lipid peroxidation, whereas ferrous sulfate, hydrogen peroxide, iron EDTA, disodium-EDTA, and β-carotene induced MDA formation. In contrast, quenchers of oxygen free radicals such as HEPES, MES, MOPS, PIPES, phenylalanine, Tiron, thiourea, sodium azide, and sodium cyanide (uncouplers of oxidative phosphorylation) caused somewhere between a 12 to 70 percnt; reduction in MDA production. TBA-reactive products were formed despite the incorporation of superoxide dismutase, proxidase, and catalase into the reaction mixture.     

Nodule-specific polypeptides from effective alfalfa root nodules and from ineffective nodules lacking nitrogenase

In addition to leghemoglobin, at least nine nodule-specific polypeptides from the alfalfa (Medicago sativa L.)-Rhizobium meliloti symbiosis were identified by immune assay. Some of these polypeptides may be subunits of larger proteins but none appeared to be subunits of the same multimeric protein. All nine of the nodule-specific polypeptides were localized to within the plant cytosol; they were not found in extracts of bacteroids or in the peribacteroid space. At least one of these nodule-specific polypeptides was found to be antigenically related to nodule-specific polypeptides in pea and/or soybean. Ineffective nodules elicited by R. meliloti strains containing mutations in four different genes required for nitrogenase synthesis contained reduced concentrations of leghemoglobin and of several of the nodule-specific polypeptides. Other nodule-specific polypeptides were unaltered or actually enriched in the ineffective nodules. Many of the differences between the ineffective and effective nodules were apparent in nodules harvested shortly after the nodules became visible. These differences were greatly amplified in older nodules. When the four ineffective nodule types were compared to one another, there were clear quantitative differences in the concentrations of several of the nodule-specific polypeptides. These differences suggest that lack of a functional nitrogenase does not have a single direct effect on nodule development.     

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.