A functional relationship between leghaemoglobin and nitrogenase based on novel measurements of the two proteins in legume root nodules

A combination of physiological and structural measurements made on nodulated cowpea and soybean plants cultured with roots in different pO(2) permitted the expression of data in various ways. Values of leghemoglobin concentration and nitrogenase activity from the two legumes were expressed conventionally either on a per plant or per gram nodule fresh weight basis, and where microscopy was done, on the basis of nitrogenase-containing, N(2)-fixing units (i.e. per bacteroid, per infected cell, or per gram infected tissue). In both legumes, acetylene reduction, N fixed and ureide content expressed on the basis of whole plants or per nitrogenase-containing units were very significantly correlated with values of leghaemoglobin concentrations expressed in a similar manner. The use of mathematical correlations in this study involving leghaemoglobin concentrations and various indices of N(2) fixation indicated a strong functional relationship between the two proteins in symbiotic legumes. These findings confirm previous suggestions that leghaemoglobin and the nitrogenase complex are two proteins closely associated with N(2)-fixing efficiency in legume root nodules.     

Effect of salt stress on nodulation and nitrogen fixation in legumes

It is now well established that almost all phases of root nodule development in legumes are adversely affected by saline conditions in the rooting medium. There is also a general agreement that the rhizobia are more tolerant to salt stress than the host plant, but they show considerable strain variability in growth and survival under saline conditions. Inhibitory effect of salinity on nodulation has been attributed to decrease in rhizobial colonisation and shrinkage and lack of root hair formation. Salt stress also induces premature senescence of already formed nodules. Both N2-fixation activity and nodule respiration are inhibited sharply on exposure of plants to saline conditions. The decrease in N2-fixation has been ascribed to direct effect on nitrogenase activity or an indirect effect through decrease in leghemoglobin content, respiratory rate, malate concentrations in nodules and photosynthate availability. Salinity increases oxygen diffusion resistance in the nodules and alters their ultrastructure. Decrease in N2-fixation in nodules under salinity is also accompanied by parallel decrease in the activity of H2O2-scavenging enzymes like catalase, ascorbate peroxidase and the level of antioxidants like ascorbic acid. Nodules appear to undergo osmoregulation under saline conditions by accumulating physiologically compatible solutes like proline, sugars (pinnitol) and lactic acid. The intensity of the adverse effects of salinity on nodule functioning depends on plant species, rhizobial strain, duration of exposure to saline conditions, nature, concentration and mode of salt application.     

The soybean NRAMP homologue,GmDMT1, is a symbiotic divalent metal transporter capable of ferrous iron transport

Iron is an important nutrient in N2-fixing legume root nodules. Iron supplied to the nodule is used by the plant for the synthesis of leghemoglobin, while in the bacteroid fraction, it is used as an essential cofactor for the bacterial N2-fixing enzyme, nitrogenase, and iron-containing proteins of the electron transport chain. The supply of iron to the bacteroids requires initial transport across the plant-derived peribacteroid membrane, which physically separates bacteroids from the infected plant cell cytosol. In this study, we have identified Glycine max divalent metal transporter 1 (GmDmt1), a soybean homologue of the NRAMP/Dmt1 family of divalent metal ion transporters. GmDmt1 shows enhanced expression in soybean root nodules and is most highly expressed at the onset of nitrogen fixation in developing nodules. Antibodies raised against a partial fragment of GmDmt1 confirmed its presence on the peribacteroid membrane (PBM) of soybean root nodules. GmDmt1 was able to both rescue growth and enhance 55Fe(II) uptake in the ferrous iron transport deficient yeast strain (fet3fet4). The results indicate that GmDmt1 is a nodule-enhanced transporter capable of ferrous iron transport across the PBM of soybean root nodules. Its role in nodule iron homeostasis to support bacterial nitrogen fixation is discussed.     

Effects of exogenous application and stem infusion of ascorbate on soybean (Glycine max) root nodules

Numerous biochemical and physiological studies have demonstrated the importance of ascorbate (ASC) as a reducing agent and antioxidant in higher plant metabolism. Of special note is the capacity of ASC to eliminate damaging activated oxygen species (AOS) including O₂^−· and H₂O₂. N₂-fixing legume nodules are especially vulnerable to oxidative damage because they contain large amounts of leghaemoglobin which produces AOS through spontaneous autoxidation; thus, ASC and other components of the ascorbate–reduced glutathione (ASC–GSH) pathway are critical antioxidants in nodules. In order to establish a meaningful correlation between concentrations of ASC and capacity for N₂ fixation in legume root nodules, soybean ( Glycine max ) plants were treated with excess ASC via exogenous irrigation or continuous intravascular infusion through needles inserted directly into plant stems. Treatment with ASC led to striking increases in nitrogenase activity (acetylene reduction), nodule leghaemoglobin content, and activity of ASC peroxidase, a key antioxidant enzyme. The concentration of lipid peroxides, which are indicators of oxidative damage and onset of senescence, was decreased in ASC-treated nodules. These results support the conclusion that ASC is critical for N₂ fixation and that elevated ASC allows nodules to maintain a greater capacity to fix N₂ over longer periods.     

Nitrogenase activity in Alnus incana root nodules. Responses to O(2) and short-term N(2) deprivation

O(2) and host-microsymbiont interactions are key factors affecting the physiology of N(2)-fixing symbioses. To determine the relationship among nitrogenase activity of Frankia-Alnus incana root nodules, O(2) concentration, and short-term N(2) deprivation, intact nodulated roots were exposed to various O(2) pressures (pO(2)) and Ar:O(2) in a continuous flow-through system. Nitrogenase activity (H(2) production) occurred at a maximal rate at 20% O(2). Exposure to short-term N(2) deprivation in Ar:O(2) carried out at either 17%, 21%, or 25% O(2) caused a decline in the nitrogenase activity at 21% and 25% O(2) by 12% and 25%, respectively. At 21% O(2), nitrogenase activity recovered to initial activity within 60 min. The decline rate was correlated with the degree of inhibition of N(2) fixation. Respiration (net CO(2) evolution) decreased in response to the N(2) deprivation at all pO(2) values and did not recover during the time in Ar:O(2). Increasing the pO(2) from 21% to 25% and decreasing the pO(2) from 21% to 17% during the decline further decreased rather than stimulated nitrogenase activity, showing that the decline was not due to O(2) limitation. The decline was possibly due to a temporary disturbance in the supply of reductant to nitrogenase with a partial O(2) inhibition of nitrogenase at 25% O(2). These results are consistent with a fixed O(2) diffusion barrier in A. incana root nodules, and show that A. incana nodules differ from legume nodules in the response of the nitrogenase activity to O(2) and N(2) deprivation.     

Interaction between hydrogenase, nitrogenase, and respiratory activities in a Frankia isolate from Alnus rubra

H2 uptake and H2-supported O2 uptake were measured in N2-fixing cultures of Frankia strain ArI3 isolated from root nodules of Alnus rubra. H2 uptake by intact cells was O2 dependent and maximum rates were observed at ambient O2 concentrations. No hydrogenase activity could be detected in NH4+-grown, undifferentiated filaments cultured aerobically indicating that uptake hydrogenase activity was associated with the vesicles, the cellular site of nitrogen fixation in Frankia. Hydrogenase activity was inhibited by acetylene but inhibition could be alleviated by pretreatment with H2. H2 stimulated acetylene reduction at supraoptimal but not suboptimal O2 concentrations. These results suggest that uptake hydrogenase activity in ArI3 may play a role in O2 protection of nitrogenase, especially under conditions of carbon limitation.     

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.     

Nitric oxide increases the enzymatic activity of three ascorbate peroxidase isoforms in soybean root nodules

Ascorbate peroxidase is one of the major enzymes regulating the levels of H₂O₂ in plants and plays a crucial role in maintaining root nodule redox status. We used fully developed and mature nitrogen fixing root nodules from soybean plants to analyze the effect of exogenously applied nitric oxide, generated from the nitric oxide donor 2,2′-(hydroxynitrosohydrazono)bis-ethanimine, on the enzymatic activity of soybean root nodule ascorbate peroxidase. Nitric oxide caused an increase in the total enzymatic activity of ascorbate peroxidase. The nitric oxide-induced changes in ascorbate peroxidase enzymatic activity were coupled to altered nodule H₂O₂ content. Further analysis of ascorbate peroxidase enzymatic activity identified three ascorbate peroxidase isoforms for which augmented enzymatic activity occurred in response to nitric oxide. Our results demonstrate that nitric oxide regulates soybean root nodule ascorbate peroxidase activity. We propose a role of nitric oxide in regulating ascorbate-dependent redox status in soybean root nodule tissue.Key words: antioxidant enzymes, ascorbate peroxidase, nitric oxide, oxidative stress, reactive oxygen species, redox homeostasis, soybean root nodules     

Purification, properties, and distribution of ascorbate peroxidase in legume root nodules

All aerobic biological systems, including N₂-fixing root nodules, are subject to O₂ toxicity that results from the formation of reactive intermediates such as H₂O₂ and free radicals of O₂. H₂O₂ may be removed from root nodules in a series of enzymic reactions involving ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. We confirm here the presence of these enzymes in root nodules from nine species of legumes and from Alnus rubra. Ascorbate peroxidase from soybean nodules was purified to near homogeneity. This enzyme was found to be a hemeprotein with a molecular weight of 30,000 as determined by sodium dodecyl sulfate gel electrophoresis. KCN, NaN₃, CO, and C₂H₂ were potent inhibitors of activity. Nonphysiological reductants such as guaiacol, o-dianisidine, and pyrogallol functioned as substrates for the enzyme. No activity was detected with NAD(P)H, reduced glutathione, or urate. Ascorbate peroxidation did not follow Michaelis-Menten kinetics. The substrate concentration which resulted in a reaction rate of ½ V_max was 70 micromolar for ascorbate and 3 micromolar for H₂O₂. The high affinity of ascorbate peroxidase for H₂O₂ indicates that this enzyme, rather than catalase, is responsible for most H₂O₂ removal outside of peroxisomes in root nodules.     

The Physiological Mechanisms Underlying N₂-Fixing Common Bean (<i>Phaseolus vulgaris</i> L.) Tolerance to Iron Deficiency

Iron is an essential element for plants as well as all living organisms, functioning in various physiological and biochemical processes such as photosynthesis, respiration, DNA synthesis, and N₂ fixation. In the soil, Fe bioavailability is extremely low, especially under aerobic conditions and at high pH ranges. In contrast, plants with nodules on their roots that fix atmospheric nitrogen need much more iron. To highlight the physiological traits underlying the tolerance of N₂-fixing common bean to iron deficiency, two genotypes were hydroponically cultivated in a greenhouse: Coco nain (CN) and Coco blanc (CB). Plants were inoculated with an efficient strain of Rhizobium tropici, CIAT899, and received a nutrient solution added with 0 µM Fe (severe Fe deficiency, SFeD), 5 µM Fe (moderate Fe deficiency, MFeD) or 45 µM Fe (control, C). Several physiological parameters related to photosynthesis and symbiotic nitrogen fixation were then analyzed. Iron deficiency significantly reduced whole plant and nodule growth, chlorophyll biosynthesis, photosynthesis, leghemoglobin (LgHb), nitrogenase (N₂ase) activity, nitrogen, and Fe nutrition, with some genotypic differences. As compared to CB, CN maintained better Fe allocation to shoots and nodules, allowing it to preserve the integrity of its photosynthetic and symbiotic apparatus, thus maintaining the key functional traits of the plant metabolism (chlorophyll biosynthesis and photosynthesis in shoots, leghemoglobin accumulation, and nitrogenase activity in root nodules). Plant growth depends on photosynthesis, which needs to be supplied with sufficient iron and nitrogen. Fe deficiency stress index (FeD-SI) and Fe use efficiency (FeUE) are two physiological traits of tolerance that discriminated the studied genotypes.     

Nodule structure and functioning in Cicer arietinum as affected by nitrate

Chickpea (Cicer arietinum L.) cv. C-235 inoculated with Rhizobium sp. (Cicer) strain cv4Az was raised in sand culture under natural conditions with nitrogen-free nutrient solution. 45-d-old plants were treated with 20 and 50 mM KNO3 and sampling made 2 and 6 d after treatment. KNO3 application induced premature nodule senescence. Light microscopic investigations showed that KNO3 treatments resulted in structural degradation of the central bacteroidal tissue. The mass of green nodules increased by 35 % under these treatments. This was accompanied by a rapid decline in leghemoglobin (Lb) content of the nodules being 51 - 67 % lower than in control. The total soluble nodule proteins showed relatively minor changes under KNO3 treatments thus suggesting preferential degradation of Lb. These changes were associated with a rapid decline in N2-fixing activity. However, the decline in total soluble sugars was relatively minor as compared to acetylene reducing activity, thus indicating that sugar deprivation is not the cause of decreased nitrogen fixation ability. Glutathione reductase and ascorbate peroxidase activity showed a 10 - 20 % decrease in comparison with the control. Accumulation of H2O2 and structural degradation of the nodular tissue are considered to be the factors leading to nodule senescence under nitrate treatments. This revised version was published online in July 2006 with corrections to the Cover Date.     

Turnover of nitrogenase and leghemoglobin in root nodules of Pisum sativum

Turnover rates of the two nitrogenase components and leghemoglobin in root nodules of pea plants nodulated with Rhizobium leguminosarum were determined with three different methods: 1, Kinetics of 35S incorporation into protein; 2, pulse-chase experiments; 3, chloramphenicol inhibition of bacteroid protein synthesis. Methods 1 and 3 revealed that the turnover rates of the two nitrogenase components and leghemoglobin are identical to the average rate of bacteroid and plant nodule protein turnover. The t1/2 times of component I and II and leghemoglobin were about 2 days. Pulse-chase experiments with 35SO(2-)4 appeared to be rather unsuitable for determination of turnover rates in pea root nodules.     

A new membrane tube technique (METT) for continuous gas measurements in soils

The metabolic activities of root nodules formed by Rhizobium tropici UM1899 were measured to test for the effects of geographical origin of the host bean (Phaseolus vulgaris L.) plant. Under increasing levels of N (0 to 24 mM of NH4NO3), the optimum condition for nitrogen fixation based on nitrogenase activity and allantoin concentration, was obtained between 2 and 4 mM N. Cultivars, including wild accessions from the two major domestication centers in America (Middle America and Andes), were then grown under aseptic conditions with 2 mM NH4NO3 and the rhizobial inoculant. Plant nodulins [leghaemoglobin (Lb), phosphoenolypyruvate carboxylase (PEPC) and glutamine synthetase (GS)], bacterial nitrogenase (NIF) activities as well as allantoin (ALA) concentration in the xylem sap, were assayed in flowering plants. Lb, PEPC, NIF activities and ALA concentrations were strongly affected by cultivar and by the center of origin. GS activity did not vary significantly with either cultivar or center of origin. LB, NIF and ALA were directly related to plant growth and offer opportunities to select for efficient N2-fixing symbioses. There were slight increases in nodulin activities of the domesticated cultivars, but the overall low variability within this material relative to landraces suggests that diversity for biological nitrogen fixation was reduced by domestication.     

Effects of ambient oxygen and of fixed nitrogen on concentrations of glutathione, ascrobate, and associated enzymes in soybean root nodules

Soybean (Glycine max [L.] Merr.) root nodules contain the enzymes of the ascorbate-glutathione cycle for defense against activated forms of oxygen. Nodulated roots of hydroponically grown soybean plants were exposed to atmospheres containing 2, 21, 50, or alternating 21 and 50 kilopascals of O₂. The activities of ascorbate (ASC) peroxidase, monodehydroascorbate (MDHA) reductase, dehydroascorbate (DHA) reductase, and glutathione (GSSG) reductase were higher in nodules exposed to high pO₂. Nodule contents of ascorbate and reduced glutathione were also greater in the high pO₂ treatments. Treatment of nodulated plants with fixed nitrogen (urea) led to concomitant decreases in acetylene reduction activity, in leghemoglobin content, and in activities of ASC peroxidase, DHA reductase, and GSSG reductase. Activity of MDHA reductase and glutathione concentrations in nodules were not affected by treatment with urea. The enzymes of the ascorbate-glutathione cycle were also detected in uninfected soybean roots, although at levels substantially below those in nodules. These observations indicate that the ascorbate-glutathione cycle can adjust to varying physiological conditions in nodules and that there is a key link between N₂ fixation and defenses against activated forms of oxygen.     

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.     

Nitrogenase Inhibition in Nodules from Pea Plants Grown Under Salt Stress Occurs at the Physiological Level and can be Alleviated by B and Ca

In order to shed new light on the mechanisms of salt-mediated symbiotic N₂-fixation inhibition, the effect of salt stress (75 mM) on N₂-fixation in pea root nodules induced by R. leguminosarum was studied at the gene expression, protein production and enzymatic activity levels. Acetylene reduction assays for nitrogenase activity showed no activity in salt-stressed plants. To know whether salt inhibits N₂-fixing activity at a molecular or at a physiological level, expression of the nifH gene, encoding the nitrogenase reductase component of the nitrogenase enzyme was analyzed by RT-PCR analysis of total RNA extracted from nodulated roots. The nifH messenger RNA was present both in plants grown in the presence and absence of salt, although a reduction was observed in salt-stressed plants. Similar results were obtained for the immunodetection of the nitrogenase reductase protein in Western-blot assays, indicating that nitrogen fixation failed mainly at physiological level. Given that nutrient imbalance is a typical effect of salt stress in plants and that Fe is a prosthetic component of nitrogenase reductase and other proteins required by symbiotic N₂-fixation, as leghemoglobin, plants were analyzed for Fe contents by atomic absorption and the results confirmed that Fe levels were severely reduced in nodules developed in salt-stressed plants. In a previous papers (El-Hamdaoui et al., 2003b), we have shown that supplementing inoculated legumes with boron (B) and calcium (Ca) prevents nitrogen fixation decline under saline conditions stress. Analysis of salt-stressed nodules fed with extra B and Ca indicated that Fe content and nitrogenase activity was similar to that of non-stressed plants. These results indicate a linkage between Fe deprivation and salt-mediated failure of nitrogen fixation, which is prevented by B and Ca leading to increase of salt tolerance.     

Influence of cadmium stress and arbuscular mycorrhizal fungi on nodule senescence in Cajanus cajan (L.) Millsp

Cadmium (Cd) causes oxidative damage and affects nodulation and nitrogen fixation process of legumes. Arbuscular mycorrhizal (AM) fungi have been demonstrated to alleviate heavy metal stress of plants. The present study was conducted to assess role of AM in alleviating negative effects of Cd on nodule senescence in Cajanus cajan genotypes differing in their metal tolerance. Fifteen day-old plants were subjected to Cd treatments--25 mg and 50 mg Cd per kg dry soil and were grown with and without Glomus mosseae. Cd treatments led to a decline in mycorrhizal infection (MI), nodule number and dry weights which was accompanied by reductions in leghemoglobin content, nitrogenase activity, organic acid contents. Cd supply caused a marked decrease in nitrogen (N), phosphorus (P), and iron (Fe) contents. Conversely, Cd increased membrane permeability, thiobarbituric acid reactive substances (TBARS), hydrogen peroxide (H2O2), and Cd contents in nodules. AM inoculations were beneficial in reducing the above mentioned harmful effects of Cd and significantly improved nodule functioning. Activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) increased markedly in nodules of mycorrhizal-stressed plants. The negative effects of Cd were genotype and concentration dependent.     

Reactive oxygen species and antioxidants in legume nodules

Reactive oxygen species are a ubiquitous danger for aerobic organisms. This risk is especially elevated in legume root nodules due to the strongly reducing conditions, the high rates of respiration, the tendency of leghemoglobin to autoxidize, the abundance of nonprotein Fe and the presence of several redox proteins that leak electrons to O₂. Consequently, nodules are particularly rich in both quantity and diversity of antioxidant defenses. These include enzymes such as superoxide dismutase (EC 1.15.1.1) and ascorbate peroxidase (EC 1.11.1.11) and metabolites such as ascorbate and thiol tripeptides. Nodule antioxidants have been the subject of intensive molecular, biochemical and functional studies that are reviewed here. The emerging theme is that antioxidants are especially critical for the protection and optimal functioning of N₂ fixation. We hypothesize that this protection occurs at least at two levels: the O₂ diffusion barrier in the nodule parenchyma (inner cortex) and the infected cells in the central zone.