Carbohydrate, organic Acid, and amino Acid composition of bacteroids and cytosol from soybean nodules

Metabolites in Bradyrhizobium japonicum bacteroids and in Glycine max (L.) Merr. cytosol from root nodules were analyzed using an isolation technique which makes it possible to estimate and correct for changes in concentration which may occur during bacteroid isolation. Bacteroid and cytosol extracts were fractionated on ion-exchange columns and were analyzed for carbohydrate composition using gas-liquid chromatography and for organic acid and amino acid composition using high performance liquid chromatography. Analysis of organic acids in plant tissues as the phenacyl derivatives is reported for the first time and this approach revealed the presence of several unknown organic acids in nodules. The time required for separation of bacteroids and cytosol was varied, and significant change in concentration of individual compounds during the separation of the two fractions was estimated by calculating the regression of concentration on time. When a statistically significant slope was found, the true concentration was estimated by extrapolating the regression line to time zero. Of 78 concentration estimates made, there was a statistically significant (5% level) change in concentration during sample preparation for only five metabolites: glucose, sucrose, and succinate in the cytosol and d-pinitol and serine in bacteroids. On a mass basis, the major compounds in bacteroids were (descending order of concentration): myo-inositol, d-chiro-inositol, alpha,alpha-trehalose, sucrose, aspartate, glutamate, d-pinitol, arginine, malonate, and glucose. On a proportional basis (concentration in bacteroid as percent of concentration in bacteroid + cytosol fractions), the major compounds were: alpha-aminoadipate (94), trehalose (66), lysine (58), and arginine (46). The results indicate that metabolite concentrations in bacteroids can be reliably determined.     

Metabolism of [13C]-labelled photosynthate in plant cytosol and bacteroids of root nodules of Glycine max

Well-nodulated soybean ( Glycine max L. Merr. cv. Akisengoku) plants were allowed to assimilate ¹³CO₂. Plant cytosol and bacteroid fractions were isolated from nodules, and the kinetics of [¹³C]-labelling of soluble carbohydrates, organic acids and amino acids were investigated.
The concentrations of all metabolites, with the exception of trehalose and 3-hydroxy-butyrate, were 10- to 1000-fold higher in plant cell cytosol than in bacteroids. The major portion of trehalose was found in bacteroids and 3-hydroxybutyrate only in bacteroids. Sucrose was most highly labelled with ¹³C in nodules, and the levels and time-course of labelling of sucrose were in good agreement with those of respired CO₂ from the nodules. The levels and time-courses of labelling of sucrose were closely similar in cytosol and bacteroids. Glucose was less labelled than sucrose and the level of labelling was consistently higher in cytosol than in bacteroids. The levels of [¹³C]-labelling of organic acids and amino acids in nodules were lower than those of sucrose and of respired CO₂. Tricarboxylic acid cycle intermediates, particularly succinate, were considerably less labelled in bacteroids than in the cytosol. All amino acids detected were also much more rapidly labelled in the cytosol. The results are discussed in relation to the utilization and possible compartmentation of carbon substrates in nodule tissues.     

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.

     

Proline accumulation inside symbiosomes of faba bean nodules under salt stress

A two‐week salt treatment (NaCl, 100 m M ) induced a 50% inhibition of acetylene reduction activity (ARA) of faba bean ( Vicia faba L. var. minor cv. Soravi) nodules, associated with a large increase in the nodule pool of amino acids. The concentration of proline in the different nodule compartments was determined after calculating their respective volumes from their areas on electron micrographs. The proline concentration exhibited a large increase, especially in the cytosol where its amount was 8‐fold enhanced under salt stress, whereas the low proline content of bacteroids was less affected. Increase of proline concentration in faba bean nodules subjected to salt stress was correlated with an enhancement of the cytosolic Δ¹‐pyrroline‐5‐carboxylate synthetase (EC 2.7.2.11 + EC 1.2.1.41; P5CS) activity. Experiments with purified symbiosome preparations showed that the greatest proline content occurred in the peribacteroid space (PBS), where proline was the most abundant amino acid, with a concentration reaching 15.3 m M under salt stress. Proline accumulation in the PBS resulted both from a diffusive transport from the host cell to the symbiosomes through the peribacteroid membrane (PBM) and from the very low rate of uptake by faba bean bacteroids. This accumulation could be partly responsible for the 1.7‐fold enlargement of the symbiosome volume observed in salt‐stressed nodules. In incubations of bacteroids, isolated from salt‐stressed or unstressed plants and supplied with O₂ by purified oxyleghemoglobin, addition of proline stimulated neither O₂ consumption nor ARA. These results were consistent with proline playing a role as osmoticum, rather than energy source for bacteroid N₂ fixation in amide‐exporting legumes such as faba bean.     

Pools of non-structural carbohydrates in soybean root nodules during water stress

The aim of this study was to examine how the pools of non-structural carbohydrates in soybean nodules are affected under water stress conditions depending on the nature of the symbiont strains with particular emphasis on the plant-borne carbohydrates sucrose and pinitol, and on trehalose, a compatible solute synthesized by the bacteroids. Soybean ( Glycine max [L.] Merr. cv. Maple Arrow) plants were inoculated with the nitrogen-fixing strains Bradyrhizobium japonicum 61-A-101 or USDA 110 spc4 and cultivated axenically under conditions in which nodules formed in an upper soil compartment while roots for water supply grew into a compartment filled with nutrient solution. When the nodules were well established (1 month post inoculation), 10% (w/v) PEG 6000 was added to the nutrient solution. This led to a slowly progressing, moderate water stress, as determined by measuring the decrease of transpiration, and to a decrease in nitrogen fixation. The pool sizes of the major non-structural nodule carbohydrates changed during progression of water stress. Sucrose, the major soluble carbohydrate in nodules of unstressed plants (2 and 4%, respectively of nodule dry weight depending on symbiont strain), strongly increased in nodules of stressed plants, reaching nearly 10% of dry weight. The activities of two major sucrose-consuming enzymes, sucrose synthase and alkaline invertase, decreased markedly in nodules of stressed plants. Starch decreased only transiently upon water stress. Pinitol, a cyclitol serving as compatible solute in many plants, increased more than 4 times, reaching about 1% of nodule dry weight during the stress. Trehalose, the major soluble carbohydrate synthesized by the bacteroids, increased in nodules colonized by USDA 110 spc4 from about 0.2 to 0.8% of nodule dry weight, while in nodules colonized by 61-A-101 it amounted to more than 1.5% of dry weight both with and without stress.     

Primary metabolites of Phaseolus vulgaris nodules

More ethanol soluble material (carbohydrate and amino nitrogen) was found in both host cell and bacteroid components of Phaseolus vulgaris nodules from plants grown at 28 W/m² than from plants grown at 7 W/m². The range of compounds identified was similar at the two irradiances. On feeding ¹⁴CO₂ to the plant tops at either irradiance the labelling patterns of carbohydrates and organic acids in the nodule host cells and bacteroids suggested that any or all of the following substances could be donated by the host to the bacteroids for general metabolism: sucrose, fructose, glucose, an unidentified carbohydrate, malic acid and an organic acid co-chromatographing with 6-phosphogluconate. Distribution and labelling patterns of nodule amino compounds were consistent with the hypothesis that ammonia is the primary product of nitrogen fixation within bacteroids, and that this ammonia is transported to host cells for assimilation, initially into glutamine and glutamate.     

Products of Dark CO(2) Fixation in Pea Root Nodules Support Bacteroid Metabolism

Products of the nodule cytosol in vivo dark [¹⁴C]CO₂ fixation were detected in the plant cytosol as well as in the bacteroids of pea (Pisum sativum L. cv “Bodil”) nodules. The distribution of the metabolites of the dark CO₂ fixation products was compared in effective (fix⁺) nodules infected by a wild-type Rhizobium leguminosarum (MNF 300), and ineffective (fix⁻) nodules of the R. leguminosarum mutant MNF 3080. The latter has a defect in the dicarboxylic acid transport system of the bacterial membrane. The ¹⁴C incorporation from [¹⁴C]CO₂ was about threefold greater in the wild-type nodules than in the mutant nodules. Similarly, in wild-type nodules the in vitro phosphoenolpyruvate carboxylase activity was substantially greater than that of the mutant. Almost 90% of the ¹⁴C label in the cytosol was found in organic acids in both symbioses. Malate comprised about half of the total cytosol organic acid content on a molar basis, and more than 70% of the cytosol radioactivity in the organic acid fraction was detected in malate in both symbioses. Most of the remaining ¹⁴C was contained in the amino acid fraction of the cytosol in both symbioses. More than 70% of the ¹⁴C label found in the amino acids of the cytosol was incorporated in aspartate, which on a molar basis comprised only about 1% of the total amino acid pool in the cytosol. The extensive ¹⁴C labeling of malate and aspartate from nodule dark [¹⁴C]CO₂ fixation is consistent with the role of phosphoenolpyruvate carboxlase in nodule dark CO₂ fixation. Bacteroids from the effective wild-type symbiosis accumulated sevenfold more ¹⁴C than did the dicarboxylic acid transport defective bacteroids. The bacteroids of the effective MNF 300 symbiosis contained the largest proportion of the incorporated ¹⁴C in the organic acids, whereas ineffective MNF 3080 bacteroids mainly contained ¹⁴C in the amino acid fraction. In both symbioses a larger proportion of the bacteroid ¹⁴C label was detected in malate and aspartate than their corresponding proportions of the organic acids and amino acids on a molar basis. The proportion of ¹⁴C label in succinate, 2-oxogultarate, citrate, and fumarate in the bacteroids of the wild type greatly exceeded that of the dicarboxylate uptake mutant. The results indicate a central role for nodule cytosol dark CO₂ fixation in the supply of the bacteroids with dicarboxylic acids.     

Effect of nitrate on the organic Acid and amino Acid composition of legume nodules

Nitrate supplied to legume plants inhibits the activity of nitrogenase in Rhizobium bacteroids in root nodules. The accumulation of amino N which is known to occur in Glycine max (L.) Merr. nodules as nitrogenase activity declines was studied in more detail by analysis of changes in free amino acid composition in response to high nitrate supply. A 6-fold increase in asparagine concentration in Bradyrhizobium japonicum bacteroids was found about the time of maximum nitrogenase inhibition. However, the accumulation of amino acids in soybean nodules lagged behind the inhibition of nitrogenase. Furthermore, in studies of a second legume, Phaseolus vulgaris (L.) inoculated with two different strains of Rhizobium phaseoli, a high nitrate treatment inhibited nitrogenase but had no significant effect on amino acid composition of nodules. The possibility that nitrate may interfere with the supply of carbon substrates to bacteroids was examined by the analysis of organic acids in legume nodules supplied with nitrate. Nitrate had a small (10-20%) negative effect on the concentration of tricarboxylic acid cycle acids in P. vulgaris nodules. However, in G. max nodules, high nitrate treatment resulted in significant increases in the concentration of malate, succinate, fumarate, and citrate. Thus, carbon deprivation of bacteroids also seems unlikely as a cause of the inhibition of nitrogenase by nitrate. There was a transient increase in ammonium concentration in P. vulgaris nodules in response to high nitrate treatment. This effect was rapid relative to other effects of nitrate on nodule composition and was roughly coincident with the rapid decline in acetylene reduction activity.     

Sugar and organic Acid constituents in white clover

Major ethanol-soluble carbohydrate and organic acid constituents of white clover (Trifolium repens) have been identified by use of high-performance liquid chromatography and gas chromatography. In leaves, petioles, roots, and nodules, pinitol (3-O-methyl chiro-inositol) is the predominant sugar, with sucrose present in lower concentration. In leaves and petioles there are significant levels of α- and β-methyl glucosides, linamarin, glucose, and fructose. In the nodules glucose is rarely present at detectable levels. The concentration of pinitol is generally greater than 25 millimolar in each tissue examined whereas the level of sucrose varies depending on the time of day. Sucrose is the major sugar significantly labeled during 1 hour administration of ¹⁴CO₂ and accounts for more than 99% of all the radioactivity detected in the nodules at early times. Between 3 and 7 hours after labeling, 6% of the radioactivity is found in the organic acids fraction and 5% in the basic fraction of nodules. Malonic acid does not appear to be present in unusually high concentrations in either leaves or nodules of white clover.     

Nodule carbohydrate metabolism and polyols involvement in the response of Medicago sativa to salt stress

Alterations of plant growth, chlorophyll fluorescence parameters, nodule carbon metabolism and polyols concentration as result of salt stress were examined in alfalfa (Medicago sativa). Plants, in symbiosis with Sinorhizobium meliloti GR4 strain, were grown under controlled conditions for 35 days (DAS) and subjected to 150 mM of NaCl stress. Plant biomass (PDW) and nitrogen fixation rate (NFR) were markedly affected by salt stress conditions; the highest reductions of PDW (50%) and NFR (40%) were registered at 84 DAS and 56 DAS, respectively. In addition, salinity affected the chlorophyll fluorescence parameters, decreased initial chlorophyll fluorescence (F₀) and increased the optimum quantum yield of PSII (F_v/F_m ratio). The enzyme activities sucrose synthase activity and phosphoenolpyruvate carboxylase, responsible for the carbon supply to the bacteroids by the formation of dicarboxylates, were drastically inhibited by salinity, mainly at 56 DAS with the beginning of flowering. The content of total soluble sugars and proline increased under salt stress, and these concentrations were higher in nodule than in leaf. This last result suggests that the nodule is an organ specially protected in order to maintain its functioning, even under stress conditions. Besides, the content of myoinositol and pinitol in leaves and nodules changed with the plant growth stage and the saline treatment. Under salinity stress, the concentrations of pinitol in nodule were higher than in leaf, which supports the central function of this molecule in the adaptive response of nodules to salt stress. The increase of pinitol synthesis in nodule of M. sativa under salt stress could be one of the adaptive features used by the plant.     

Phloem‐derived γ‐aminobutyric acid (GABA) is involved in upregulating nodule N2 fixation efficiency in the model legume Medicago truncatula

Nitrogen fixation in legumes is downregulated through a whole plant N feedback mechanism, for example, when under stress. This mechanism is probably triggered by the impact of shoot‐borne, phloem‐delivered compounds. However, little is known about any whole‐plant mechanism that might upregulate nitrogen fixation, for example, under N deficiency. We induced emerging N‐deficiency through partial excision of nodules from Medicago truncatula plants. Subsequently, the activity and composition of the remaining nodules and shifts in concentration of free amides/amino acids in the phloem were monitored. Furthermore, we mimicked these shifts through artificial feeding of γ‐aminobutyric acid (GABA) into the phloem of undisturbed plants. As a result of increased specific activity of nodules, N₂ fixation per plant recovered almost completely 4–5 d after excision. The concentration of amino acids, sugars and organic acids increased strongly in the upregulated nodules. A concomitant analysis of the phloem revealed a significant increase in GABA concentration. Comparable with the effect of nodule excision, artificial GABA feeding into the phloem resulted in an increased activity and higher concentration of amino acids and organic acids in nodules. It is concluded that GABA might be involved in upregulating nodule activity, possibly because of its constituting part of a putative amino acid cycle between bacteroids and the cytosol.     

Salt tolerance of nitrogen fixation in <Emphasis Type="Italic">Medicago ciliaris</Emphasis> is related to nodule sucrose metabolism performance rather than antioxidant system

In this study, the effect of 100 mM NaCl on physiological and biochemical responses were investigated in nodules of two Medicago ciliaris lines differing in salt tolerance (TNC 1.8 and TNC 11.9). Results showed that, on the basis of growth and nitrogen fixation, the line TNC 1.8 proved more salt tolerant than TNC 11.9. The salt-induced oxidative stress (membrane lipid peroxidation, leghemoglobin degradation, antioxidant activities reduction) occurred similarly in nodules of both lines. The tolerant line TNC 1.8 showed a better capacity to preserve higher sucrolytic activities and maintained higher nodule malate concentration, although total organic acids decreased in both lines. The higher amount of organic acids in the tolerant line seems to be related to its capacity to maintain higher NH₄ nodule concentration in comparison with the sensitive line. Although salt stress reduced concentrations of the majority of amino acid in both lines, the decrease of the most preponderant amino acids glycine, valine, aspartate and glutamate was more accentuated in the sensitive line TNC 11.9. However, alanine concentration increased in the nodules of this sensitive line, suggesting a higher incidence of stress-induced hypoxia. The present study provides further evidence that salt tolerance of nitrogen fixation in the tolerant line is linked to a more effective supply of malate to bacteroids which allows the synthesis of amino acids required to maintain both plant and nodule growth.     

Effects of salt stress on growth, photosynthesis and nitrogen fixation in chick-pea (Cicer arietinum L.)

Plants of chick-pea (Cicer arietinum L. cv. ILC1919) inoculated with Mesorhizobium ciceri strain ch-191 were grown in a controlled environmental chamber, and were administered salt (0, 50, 75, and 100 mM NaCl) during the vegetative period. Four harvests (4, 7, 11, and 14d after treatment) were analysed. The aim was to ascertain whether the negative effect of saline stress on nitrogen fixation is due to a limitation on the photosynthate supply to the nodule or a limitation on the nodular metabolism which sustains nitrogenase activity.Plant growth was affected only by the highest NaCl concentration, whereas nitrogenase activity was affected from 50 mM. At the first harvest, Rubisco, PEPC and MDH activities in leaves rose with salt, but fell during the following harvests. The increase of PEPC and MDH in nodules at the two first samplings was clearly related to salt concentration. While 50 mM NaCl increased GS and GOGAT in nodules at some harvests, 100 mM strongly inhibited these activities at all the harvests. The accumulation of proline, amino acids and carbohydrates was clearly related to salt especially in the leaves, whereas in the nodules the protein content was boosted by salt. Although photosynthesis declined with NaCl, the response of nitrogen fixation to salt was more pronounced. This situation, together with carbohydrate accumulation, suggests that the lack of photosynthate does not cause the inhibition of nitrogenase activity under this type of stress. The similar trend observed for the PEPC-MDH pathway and the ARA support the hypothesis concerning the limitation in the supply of energy substrate, mainly malate, to the bacteroids. The accumulation of compatible solutes is more a consequence of damage produced by salt stress than of a protective strategy.     

Proline betaine accumulation and metabolism in alfalfa plants under sodium chloride stress. Exploring its compartmentalization in nodules

The osmoprotectant Pro betaine is the main betaine identified in alfalfa (Medicago sativa). We have investigated the long-term responses of nodulated alfalfa plants to salt stress, with a particular interest for Pro betaine accumulation, compartmentalization, and metabolism. Exposure of 3-week-old nodulated alfalfa plants to 0.2 m NaCl for 4 weeks was followed by a 10-, 4-, and 8-fold increase in Pro betaine in shoots, roots, and nodules, respectively. Isotope-labeling studies in alfalfa shoots indicate that [14C]Pro betaine was synthesized from l-[14C]Pro. [14C]Pro betaine was efficiently catabolized through sequential demethylations via N-methylPro and Pro. Salt stress had a minor effect on Pro betaine biosynthesis, whereas it strongly reduced Pro betaine turnover. Analysis of Pro betaine and Pro compartmentalization within nodules revealed that 4 weeks of salinization of the host plants induced a strong increase in cytosol and bacteroids. The estimated Pro betaine and Pro concentrations in salt-stressed bacteroids reached 7.4 and 11.8 mm, respectively, compared to only 0.8 mm in control bacteroids. Na+ content in nodule compartments was also enhanced under salinization, leading to a concentration of 14.7 mm in bacteroids. [14C]Pro betaine and [14C]Pro were taken up by purified symbiosomes and free bacteroids. There was no indication of saturable carrier(s), and the rate of uptake was moderately enhanced by salinization. Ultrastructural analysis showed a large peribacteroid space in salt-stressed nodules, suggesting an increased turgor pressure inside the symbiosomes, which might partially be due to an elevated concentration in Pro, Pro betaine, and Na+ in this compartment.     

Estimation of ammonium concentration in the cytosol of soybean nodules

Analysis of ammonium concentration in the cytosol of soybean (Glycine max [L.] Merr.) root nodules gave high levels of error variation. When the separation of cytosol and bacteroids was deliberately delayed following nodule maceration, a large increase in ammonium concentration was found in the cytosol. When a series of samples was subjected to delay intervals of 0 to 60 minutes, extrapolation of the regression line to time zero indicated that the ammonium concentration in cytosol at the time of nodule maceration was essentially nil. The source of ammonium buildup following maceration was not found, but hydrolysis of free amino acids or ureides was ruled out. An extremely low concentration of ammonium in the cytosol is consistent with a model for movement of ammonia (or ammonium) from bacteroids to host cytoplasm by diffusion.     

Metabolite profiles of nodulated alfalfa plants indicate that distinct stages of nodule organogenesis are accompanied by global physiological adaptations

An effective symbiosis between Sinorhizobium meliloti and its host plant Medicago sativa is dependent on a balanced physiological interaction enabling the microsymbiont to fix atmospheric nitrogen. Maintenance of the symbiotic interaction is regulated by still poorly understood control mechanisms. A first step toward a better understanding of nodule metabolism was the determination of characteristic metabolites for alfalfa root nodules. Furthermore, nodules arrested at different developmental stages were analyzed in order to address metabolic changes induced during the progression of nodule formation. Metabolite profiles of bacteroid-free pseudonodule extracts indicated that early nodule developmental processes are accompanied by photosynthate translocation but no massive organic acid formation. To determine metabolic adaptations induced by the presence of nonfixing bacteroids, nodules induced by mutant S. meliloti strains lacking the nitrogenase protein were analyzed. The bacteroids are unable to provide ammonium to the host plant, which is metabolically reflected by reduced levels of characteristic amino acids involved in ammonium fixation. Elevated levels of starch and sugars in Fix(-) nodules provide strong evidence that plant sanctions preventing a transformation from a symbiotic to a potentially parasitic interaction are not strictly realized via photosynthate supply. Instead, metabolic and gene expression data indicate that alfalfa plants react to nitrogen-fixation-deficient bacteroids with a decreased organic acid synthesis and an early induction of senescence. Noneffective symbiotic interactions resulting from plants nodulated by mutant rhizobia also are reflected in characteristic metabolic changes in leaves. These are typical for nitrogen deficiency, but also highlight metabolites potentially involved in sensing the N status.     

Utilization of aldehydes and alcohols by soybean bacteroids

Aldehydes, alcohols and acids were tested for their ability to support acetylene reduction and oxygen consumption by Rhizobium japonicum bacteroids isolated from soybean nodules. Several alcohols and aldehydes increased acetylene reduction and oxygen uptake. This is consistent with the concept that the plant nodule cytosol can metabolize carbohydrate via anaerobic fermentative pathways.     

Effects of Oxygen Concentration and Leghemoglobin on Organic Acid Degradation by Isolated Soybean Nodule Bacteriods

Bacteroids retaining high acetylene reduction activity (nitrogenase activity) were prepared anaerobically from soybean nodules. Addition of succinate (or of both leghemoglobin and succinate) to the acetylene reduction assay system greatly increased the activity of the isolated bacteroids.

When various organic acids were incubated with the bacteroids at 2% oxygen concentration, an optimum condition for bacteroid acetylene reduction, the organic acid degradation by bacteroids was very slow, and both lactate and acetate were accumulated in the incubation system, suggesting the operation of fermentative pathway in bacteroids under such low oxygen conditions.

With 20% oxygen, the added organic acids were degraded rapidly by bacteroids without addition of leghemoglobin to the incubation system.

With leghemoglobin in the incubation system, the organic acid degradation by bacteroids was accelerated extensively even at 2% oxygen, and the formation of lactate and acetate were negligible. No significant difference in the organic acid degradation rate was observed between the 2% and 20% oxygen concentrations when the leghemoglobin was present in the incubation system. Addition of acetylene to the assay system slightly inhibited the organic acid degradation.

This data suggests that bacteroids are unable to oxidize organic acid in low oxygen concentration and that the leghemoglobin allows the rapid organic acid dagradation by bacteroids even in such low oxygen concentrations.     

Effect of Salt Stress on Carbon Metabolism and Bacteroid Respiration in Root Nodules of Common Bean (Phaseolus vulgaris L.)

Abstract: In the present work, we examined the effect of salinity on growth, N fixation and carbon metabolism in the nodule cytosol and bacteroids of Phaseolus vulgaris, and measured the O₂ consumption by bacteroids incubated with or without the addition of exogenous respiratory substrates. The aim was to ascertain whether the compounds that accumulate under salt stress can increase bacteroid respiration and whether this capacity changes in response to salinity in root nodules of Phaseolus vulgaris. The plants were grown in a controlled environment chamber, and 50, 100 mM or no NaCl (control) was added to the nutrient solution. Two harvests were made, at the vegetative growth period and at the beginning of the reproductive period. The enzyme activities in the nodule cytosol were reduced by the salt treatments, while in the bacteroid cytosol the enzyme activities increased at high salt concentrations at the first harvest and for ADH in all treatments. The data presented here confirm that succinate and malate are the preferred substrates for bacteroid respiration in common bean, but these bacteroids may also utilize glucose, either in control or under saline conditions. The addition of proline or lactate to the incubation medium significantly raised oxygen consumption in the bacteroids isolated from plants treated with salt.