Intact Soybean Nodules Research Articles

Effectiveness of ascorbate and ascorbate peroxidase in promoting nitrogen fixation in model systems

Ascorbate and ascorbate peroxidase are important antioxidants that are abundant in N 2-fixing legume root nodules. Antioxidants are especially critical in root nodules because leghemoglobin, which is present at high concentrations in nodules, is prone to autoxidation and production of activated oxygen species such as O 2 − and H 2O 2. The merits of ascorbate and ascorbate peroxidase for maintaining conditions favorable for N 2 fixation were examined in two model systems containing oxygen-binding proteins (purified myoglobin or leghemoglobin) and N 2-fixing microorganisms (free-living Azorhizobium or bacteroids of Bradyrhizobium japonicum) in sealed vials. The inclusion of ascorbate alone to these systems led to enhanced oxygenation of hemeproteins, as well as to increases in nitrogenase (acetylene reduction) activity. The inclusion of both ascorbate and ascorbate peroxidase resulted in even greater positive responses, including increases of up to 4.5-fold in nitrogenase activity. In contrast, superoxide dismutase did not provide beneficial antioxidant action and catalase alone provided only very marginal benefit. Optimal concentrations were 2 mM for ascorbate and 200 μg/ml for ascorbate peroxidase. These concentrations are similar to those found in intact soybean nodules. These results support the conclusion that ascorbate and ascorbate peroxidase are beneficial for maintaining conditions favorable for N 2 fixation in nodules.

Direct Detection of Radicals in Intact Soybean Nodules: Presence of Nitric Oxide-Leghemoglobin Complexes

Electron paramagnetic resonance spectroscopy has been employed to examine the nature of the metal ions and radicals present in intact root nodules of soybean plants grown in the absence of nitrate. The spectra obtained from nodules of different ages using this non-invasive technique show dramatic differences, suggesting that there are both qualitative and quantitative changes in the metal ion and radical species present. A major component of the spectra obtained from young nodules is assigned to a complex (Lb-NO) of nitric oxide (NO •) with the heme protein leghemoglobin (Lb). This Lb-NO species, which has not been previously detected in intact root nodules of plants grown in the absence of nitrate, is thought to be formed by reaction of nitric oxide with iron(II) leghemoglobin. The nitric oxide may be generated from arginine via a nitric oxide synthase-like activity present in the nodules of the soybean plants, in a manner analogous to that recently described for Lupinus albus. This Lb-NO complex is present at lower concentrations in older nodules, and is almost completely absent from senescent nodules. Exposure of young and mature nodules to oxidant stress, in the form of hydrogen peroxide, results in changes in the EPR spectra, with the loss of the signals from the Lb-NO complex and appearance of absorptions similar to those from untreated senescent nodules. These results suggest that there are characteristic changes in both the metal ion complexes and radicals present in intact root nodules of different ages, and support the theory that nitric oxide and other radicals play a significant role in determining the nitrogen fixing activity of root nodules; the modulatory activity of NO • may involve regulation of gene activity.

In Vivo Regulatory Phosphorylation of Soybean Nodule Phosphoenolpyruvate Carboxylase

In this report we provide evidence that cytosolic phosphoenolpyruvate carboxylase (PEPC) in soybean (Glycine max L.) root nodules is regulated in vivo by a seryl-phosphorylation cycle, as with the C4, Crassulacean acid metabolism, and C3 leaf isoforms. Pretreatment of parent plants by stem girdling for 5 or 14 h caused a significant decrease in the apparent phosphorylation state of nodule PEPC, as indicated by the 50% inhibition constant (L-malate) and specific activity values assayed at suboptimal conditions, whereas short-term darkness alone was without effect. However, extended (26 h) darkness led to the formation of a relatively dephosphorylated nodule PEPC, an effect that was reversed by illuminating the darkened plants for 3 h. This reversal of the apparent phosphorylation state in the light was prevented by concomitant stem girdling. In contrast, the optimal activity of nodule PEPC and its protein level showed little or no change in all pretreated plants. These results suggest that the phosphorylation state of PEPC in soybean root nodules is possibly modulated by photosynthate transported recently from the shoots. In situ [32P]orthophosphate labeling, immunoprecipitation, and phosphoamino acid analyses confirmed directly that PEPC in detached intact soybean nodules is phosphorylated on a serine residue(s).

Structural changes in the innercortex cells of soybean root nodules are induced by short‐term exposure to high salt or oxygen concentrations

ABSTRACTAfter a 2 h exposure of intact soybean nodules to high concentrations of NaCl (100mol m−3) or oxygen (8OkPa O2), morphometric computations carried out using an image analysis technique on semi‐thin sections showed that both treatments induced a decrease in the area of the inner‐cortex cells, which were then characterized by a tangential elongation. In contrast, no significant change in area occurred in the middle‐cortex cells although their elongation decreased. Electron microscopic observations showed that in the inner‐cortex cells changes included the presence of wall infoldings, an enlarged periplasmic space and a lobate nucleus whose chromatin distribution differed from that of the control. Structural changes also occurred in the endoplasmic reticulum, microbodies, mitochondria and plastids. From several of these changes, which are similar to those noted in osmocontractil cells in response to external stimuli, it can be hypothesized that the inner cortex may provide a potential mechanism for the control of oxygen diffusion through the nodules.

Characteristics of Asparagine Pool in Soybean Nodules in Comparison with Ureide Pool

The changes in the content of nitrogenous compound in intact soy bean nodules were investigated by inhibiting nitrogen fixation with acetylene application. The contents of ureides (allantoin and allantoic acid), asparagine, and glutamine decreased by the acetylene treatment. Incorporation of 15N into asparagine, ureides, and other nitrogenous compounds in intact soybean nodules was also investigated by the long term feeding of gaseous 15N2. Approximately 68% nitrogen in the ureide pool and 35% nitrogen in the asparagine pool were derived from gaseous 15N2 at 5.5 h after 15N2 exposure. The flow rate of newly fixed nitrogen via the asparagine pool was from 17 to 22% of that via the ureide pool, which was estimated based on the data of the acetylene inhibition experiment and 15N tracer experiment. A larger quantity of UN was incorporated into the amido-N of asparagine than into its amino-N, and 15N incorporation into ammonia was almost similar to that into asparagine at 5.5 h after 15N2 exposure. The charact...

Entry of Oxygen and Nitrogen into Intact Soybean Nodules

The apparent nitrogenase activity of soybean root nodules is reduced approximately 70% upon nodule detachment. By increasing the partial pressure of oxygen in the assay vessel from approximately 0 2 to 0 5 atm, the nitrogenase activity of detached nodules is restored to the original attached level. On the other hand, maximal assayable nitrogenase activity of nodules attached to the intact soybean plant is achieved in the presence of 0-2-0-3 atm of oxygen. Exposure of attached nodules to 0-5 atm oxygen causes a decrease in nitrogenase activity seemingly due to inactivation of nitrogenase. These and other data suggest that, upon detachment, the entry of oxygen into the nodules is impaired. A model for the regulation of airflow into attached and detached nodules is presented. This model is also consistent with response of nodule activity to water stress.

Incorporation of15N into various nitrogenous compounds in intact soybean nodules after exposure to15N2gas

Abstract The intact nodules attached to the upper part of soybean roots were exposed to 15N2 and the incorporation of 15N into various soluble nitrogen constituents was investigated. Results indicated that ammonia, a primary product of N2 fixation, was located in more than two compartments. Ammonia reduced from N2 gas seemed to be incorporated firstly into glutamine especially amido-group nitrogen. Newly fixed nitrogen was secondly incorporated into glutamic acid and alanine in this sequence. These results suggested that fixed ammonia was assimilated by glutamine synthetase/glutamate synthase pathway. Turn-over rate of allantoin plus allantoic acid and serine was relatively high, although apparently these compounds were not primary products of newly fixed ammonia. 15N content of allantoin was always higher than that of allantoic acid. 15N incorporation to aspartic acid and asparagine was relatively slow, especially in early period. In bacteroid fraction there is much amount of ammonia comparing with other compounds, while allantoin and asparagine were presented exclusively in cytosol. 15N was incorporated into nitrate within a few minutes especially in bacteroids.

Determination of Hydrogenase in Free-living Cultures of Rhizobium japonicum and Energy Efficiency of Soybean Nodules

A sensitive tritium exchange assay was applied to the Rhizobium system for measuring the expression of uptake hydrogenase in free-living cultures of Rhizobium japonicum. Hydrogenase was detected about 45 hours after inoculation of cultures maintained under microaerophilic conditions (about 0.1% O(2)). The tritium exchange assay was used to screen a variety of different strains of R. japonicum (including major production strains) with the findings that about 30% of the strains expressed hydrogenase activity with identical results being observed using an alternative assay based on uptake of H(2). The relative efficiency of intact soybean nodules inoculated with 10 different rhizobial strains gave results identical to those obtained using free-living cultures. The tritium exchange assay provides an easy, quick, and accurate assessment of H(2) uptake efficiency of intact nodules.

Adaptation of Nitrogen Fixation by Intact Soybean Nodules to Altered Rhizosphere pO2

The N(2)-fixing legume nodule requires O(2) for ATP production; however, the O(2) sensitivity of nitrogenase dictates a requirement for a low pO(2) inside the nodule. The effects of long term exposures to various pO(2)s on N(2)[C(2)H(2)] fixation were evaluated with intact soybean (Glycine max [L.] Merr., var. Wye) plants. Continuous exposure of their rhizosphere to a pO(2) of 0.06 atmospheres initially reduced nitrogenase activity by 37 to 45% with restoration of original activity in 4 to 24 hours and with no further change in tests up to 95 hours; continuous exposure to 0.02 atmosphere of O(2) initially reduced nitrogenase activity 72%, with only partial recovery by 95 hours. Similar exposures to a pO(2) of 0.32 atmospheres had little effect on N(2)[C(2)H(2)] fixation; a pO(2) of 0.89 atmospheres initially reduced nitrogenase activity by 98% with restoration to only 14 to 24% of that of the ambient O(2) controls by 95 hours. Re-exposure to ambient pO(2) of plants adapted to nonambient pO(2)s reduced N(2)[C(2)H(2)] fixation to similar magnitudes as the reductions which occurred upon initial exposure to variant pO(2) conditions, and a time period was required to readapt to ambient O(2). It is concluded that the N(2)[C(2)H(2)]-fixing system of intact soybean plants is able to adapt to a wide range of external pO(2)s as probably occur in soil. We postulate that this occurs through an undefined mechanism which enables the nodule to maintain an internal pO(2) optimal for nitrogenase activity.

Studies of the physiological role of leghaemoglobin in soybean root nodules

Evolution of H 2 by nitrogenase in intact soybean nodules was consistently inhibited by CO when the nodules were equilibrated with argon-CO mixtures for 1 h prior to adding O 2 to initiate the reaction. Evolution of H 2 by nitrogenase in bacteroid suspensions prepared from nodules, was not inhibited by CO. Dense, slowly shaken suspensions of bacteroids, with 12% O 2 in the gas phase maintained slow rates of H 2 evolution and acetylene reduction for up to 12 h. Addition of leghaemoglobin to these assays greatly enhanced the nitrogenase-mediated reactions. CO prevented stimulation by leghaemoglobin of H 2 evolution by bacteroids. Stimulation of acetylene reduction by bacteroid suspensions was dependent upon leghaemoglobin concentration up to about 1 mM. Increased shaking rates gave greater rates of acetylene reduction and O 2 uptake. Stimulation of nitrogenase activity in bacteroid suspensions by leghaemoglobin was much greater than stimulation of O 2 uptake. Increasing acetylene reduction in response to increasing agitation was accompanied by increasing oxygenation of the leghaemoglobin. The significance of these results in relation to the physiological role of leghaemoglobin in symbiotic N 2 fixation by legume root nodules is discussed.

Symbiotic Effectiveness and N2 Fixation in Nodulated Soybean

Highly efficient nitrogen fixation by nodulating bacteria is required for maximum production by leguminous plants. The symbiotic relationship between nodulating bacteria and legumes in nodule formation and nitrogen fixation is complex and variable (2). The Rhizobium and legume genotypes (strains and varieties) exert influences which determine the growth response of the host (3,4, 10, 11). The growth response may be poor or moderate to luxuriant, and these symbioses are referred to as ineffective and effective, respectively. Three ranges of effectiveness based on nitrogen content were used by Parker and Allen (12) to compare the growth response of clover inoculated with 35 strains of R. trifolii. Abel and Erdman (1) evaluated the response of field grown Lee soybeans to different strains of R. japonicum and concluded that some symbioses exhibited more effectiveness than others in increasing seed yield, protein percentage of seed, nodulation,, plant appearance and fresh plant weight. Effectiveness in nodulated soybean has not been compared with activity of the nitrogen fixing enzyme. Nitrogen fixing activity ini nodulated leguminous plants can be estimated rapidly and conveniently by the acetylene reduction assay using gas chromatographic techniques (7). Requirements for the reduction of acetylene and molecular nitrogen have been shown to be the same for intact soybean nodules (9) and for partially purified cell-free extracts of bacteroids (8). Using this assay would indirectly determine nitrogenase activity for particular soybeanRhizobium symblioses exhibiting ineffectiveness or effectiveness. The present report provides evidence that the interaction of soybean and R. japonicum in some way controls nitrogenase activity, which results in a particular growth response in the plant.

Reduction of acetylene and nitrogen gas by breis and cell-free extracts of soybean root nodules.

Cell-free extracts of Clostridiumit puisteurian uii (8) and of Azotobacter vinelandii (2) in the presence of an ATP generating system and an appropriate electron donor, catalyze the reduction of N., gas to ammonia. In recent investigations (3,4,9) of the properties of nitrogenase from these organisms, it has been established that the enzyme system is non-specific for electron acceptor and will catalyze the redtuction of nitrous oxide, cyanide. azide and acetylene in addition to N.. No reproduicible procedutres have beeni reported for the preparation from leguime nlodules of cell-free extracts capable of catalyzing thc reduiction of N., gas. Bergerson (1) has described a methodl for the demoiistratioli of nitrogen-fixation with a brei from soybeani root 11o(Ill1es. In his procedulre, strictly anaerobic conl(litions were necessary for the preparation of the brei and the sensitive '5N technique was uised for the detection of N. fixation. In most of the experiments no more than 6 m,u atoms of N were fixed per mg of brei protein. We have approached the problem of nitrogen fixation by noduiles of symbionts by measturing the rate of reduction of acetylene to ethylene. Intact soybean nodules were shown to catalyze the reduction of acetylene to ethylene at a rapid rate and the conditions Qptinium for the reduction determined (5). The purpose of this commtunication is to report conditions necessary for catalysis of acetylene reduction by breis and cell-free extracts of nodules and the catalysis of No reduction to ammonia by a cell-free extract of a nodule bacteroid preparation. Soybean plants were cultured in the greenhouse or in growth chambers and supplied with a nitrogenfree nutrient solution as described previously (5). When plants were 36 to 40 days old, nodutles were harvested, washed with tap and distilled water and tutilized for the various experiments. The production of ethylene from acetylene was assayed by a gas chromatographic procedure (5) and the reduction of N2 gas to ammonia was determined by the method of Mortenson (7). Whole soybean root nodules consistently catalyze the reduction of acetylene to ethylene (5) btit nodtules macerated uinder aerobic or anaerobic conditions in buffers of variotus types catalyze little or no acetylene reduction. The possibility that phenolic compounds might inhibit enzymes in nodile extracts was suggested by the observation that a DEAE coluimn removed large quantities of green to brown pigments from nodule extracts. These observations led to an 'investigation of procedulres f r the removal of phenolics or related compouinds from no(dtule extracts. Loomis anid Battaile (6) have showu that a solid preparationi of polvviniylpyrroli(loiie (Polyclar AT from the Genieral Aniilinie Corp.), (PVP) effectively remove(l pheniolic comiipotunids from planit extracts prepare(l in a bufferedl ascorbate solution and thuis preventted pheniolic oxidation products from inactivating certain plant enzymes. These proceduires (6) were incorporated into a method for the preparation of nodulle extracts for tuse in acetylene and N. redutction experiments. In a typical experiment 100 g of soybean nodules were collected, washed, and placed in a glove bag which was evaculated and flutshed with argon 3 times. The nodules were macerated in 150 ml of 20 mm potassium phosphate butffer (pH 7.2) containing 1 mM MgCI2 and 200 mm ascorbate adjusted to pH 7.0 with KOH. The macerate was squeezed through cheese cloth and mixed with 50 g of acid-washed PVP. After 10 minutes the PVP and macerate was squeezed through 100-mesh bolting cloth and the resulting brei utilized for the experiment described in table I. For the preparation of cell-free extracts the brei, maintained tinder anaerobic conditions, was centrifuged at 34,000 X g for 15 minuites, the supernatant discarded and the bacteroid fraction suspended in a volume of either 20 mm potassitum phosphate or 50 mm potassitum cacodylate buffer at pH 7.2, (tables II and III) equal to the volume of bacteroid pellet. The suispension was placed in a French press under an atmosphere of argon, cells were forced through