The effect of excision on O2 diffusion and metabolism in soybean nodules

Nitrogen-fixing nodules of soybean [Glycine max (L.) Merr. cv. Maple Arrow inoculated with Bradyrhizobium japonicum USDA 16] were studied before and after excision from the root to determine the role the O₂ regulation plays in the inhibition of nodule activity and the potential for using excised nodules nodules in studies of nodule metabolism. Relative nitrogenase (EC 1.7.99.2) activity (H₂ evolution in N₂:O₂) and nodule respiration (CO₂ evolution) were monitored first in intact nodulated roots and then in freshly excised nodules of the same plant to determine the time course of the decline in nodule metabolism. Folowing excision, nitrogenase activity and respiration declined rapidly in the first minute and then more gradually. After 40 min the rate of H₂ evolution was only 14–28% of that in the intact plant. In some nodules activity declined steadily, and in others there was a partial recovery in activity ca 10 min after detachment. Infected cell O₂ concentration (O_i), measured by a spectro-photometric technique, also declined after nodule detachment with a time course similar to the declines in nitrogenase activity and respiration. Following excision, O_i levels declined rapidly from ca 21 nM in attached nodules to 8–12 nM at 4–10 min after excision and then more gradually to 2–3 nM O₂ at 30–40 min after excision. These results show that the nodules' permeability to gas diffusion continued to be regulated for up to 40 min after detachement. At 40 min after detachment, when excised nodules displayed steady-state rates of gas exchange, linear increases in pO₂ from 20 to 100% at 4% min^?1 resulted in recoveries of H² and CO² evolution, indicating that O_i limited nitrogenase activity durig this period, and that energy reserves were greatly in excess of the O₂ available for respiration. When detached nodules were equilibrated for 12 h at 20, 30 and 50% O₂, O_i values measured at supra-ambient pO₂ were greater than those at 20% O₂ and were linked with a more rapid decline in nitrogenase activity. Also, increases in external pO₂ (O_c) failed to stimulate nodule metabolism, suggesting that the nodules' energy reserves were no longer greatly in excess of their respiratory demands. It was concluded that soybean nodules could provide useful material for steady-state studies of nodule metabolism between 40 and 240 min after detachment, but to attain metabolic rates equivalent to in vivo rates the nodules must be exposed to above-ambient pO₂.     

The Glycine-Glomus-Bradyhizobium symbiosis. VIII. Phosphorususe efficiency of CO2 and N2 fixation in mycorrhizal soybean

Soybean [ Glycine max (L.) Merr. cv. Hobbit] plants nodulated by Bradyhizobium japonicum strain USDA 110 were grown in pot cultures in severely P- and N-deficient soil and either colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae (Nicol. & Gerd.) Gerd. and Trappe or fertilized with a high (HP) or low (LP) level of KH₂PO₄ (0.6 or 0.3 m M , respectively), After 7 weeks of growth, nodule and chloroplast activities (C₂H₂ reduction and CO₂ exchange rate) were determined. Photosynthetic P-use efficiency of CO₂ fixation was significantly higher in VAM than in HP plants, while that of nitrogenase activity was lower. The LP plants were intermediate in both respects. The ratio of nodule to chloroplast activity [mol C₂H₂ reduced (mol CO₂ fixed)⁻¹] was highest in HP and lowest in VAM plants. Root colonization by the VAM fungus significantly increased nodule number and dry weight and reduced nodule specific mass and activity in comparison to HP plants. In spite of lower nodule activity, VAM plants were significantly larger and had higher N concentrations than the HP plants. The results suggest nonnutritional. VAM-elicited and host-mediated effects on the symbiotic functions of the legume association.     

Soybean nodulation and N2 fixation response to drought under carbon dioxide enrichment

The combined effects of carbon dioxide (CO₂) enrichment and water deficits on nodulation and N₂ fixation were analysed in soybean [Glycine max (L.) Merr.]. Two short-term experiments were conducted in greenhouses with plants subjected to soil drying, while exposed to CO₂ atmospheres of either 360 or 700 μmol CO₂ mol^–1. Under drought-stressed conditions, elevated [CO₂] resulted in a delay in the decrease in N₂ fixation rates associated with drying of the soil used in these experiments. The elevated [CO₂] also allowed the plants under drought to sustain significant increases in nodule number and mass relative to those under ambient [CO₂]. The total non-structural carbohydrate (TNC) concentration was lower in the shoots of the plants exposed to drought; however, plants under elevated CO₂ had much higher TNC levels than those under ambient CO₂. For both [CO₂] treatments, drought stress induced a substantial accumulation of TNC in the nodules that paralleled N₂ fixation decline, which indicates that nodule activity under drought may not be carbon limited. Under drought stress, ureide concentration increased in all plant tissues. However, exposure to elevated [CO₂] resulted in substantially less drought-induced ureide accumulation in leaf and petiole tissues. A strong negative correlation was found between ureide accumulation and TNC levels in the leaves. This relationship, together with the large effect of elevated [CO₂] on the decrease of ureide accumulation in the leaves, indicated the importance of ureide breakdown in the response of N₂ fixation to drought and of feedback inhibition by ureides on nodule activity. It is concluded that an important effect of CO₂ enrichment on soybean under drought conditions is an enhancement of photoassimilation, an increased partitioning of carbon to nodules and a decrease of leaf ureide levels, which is associated with sustained nodule growth and N₂ rates under soil water deficits. We suggest that future [CO₂] increases are likely to benefit soybean production by increasing the drought tolerance of N₂ fixation.     

O2 regulation and O2‐limitation of nitrogenase activity in root nodules of pea and lupin

The gas exchange characteristics of intact attached nodulated roots of pea (Pisum sativum cv. Finale X) and lupin (Lupinus albus cv. Ultra) were studied under a number of environmental conditions to determine whether or not the nodules regulate resistance to oxygen diffusion. Nitrogenase activity (H₂ evolution) in both species was inhibited by an increase in rhizosphere pO₂ from 20% to 30%, but recovered within 30 min without a significant increase in nodulated root respiration (CO₂ evolution). These data suggest that the nodules possess a variable barrier to O₂ diffusion. Also, nitrogenase activity in both species declined when the roots were either exposed to an atmosphere of Ar:O₂ or when the shoots of the plants were excised. These declines could be reversed by elevating rhizosphere pO₂, indicating that the inhibition of nitrogenase activity resulted from an increase in gas diffusion resistance and consequent O₂-limitation of nitrogenase-linked respiration. These results indicate that nodules of pea and lupin regulate their internal O₂ concentration in a manner similar to nodules of soybean, despite the distinct morphological and biochemical differences that exist between the nodules of the 3 species. Experiments in which total nitrogenase activity (TNA = H₂ production in Ar:O₂) in pea and lupin nodules was monitored while rhizosphere pO₂ was increased gradually to 100%, showed that the resistance of the nodules to O₂ diffusion maintains nitrogenase activity at about 80% of its potential activity (PNA) under normal atmospheric conditions. The O₂-limitation coefficient of nitrogenase (OLC_N= TNA/PNA) declined significantly with prolonged exposure to Ar:O₂ or with shoot excision. Together, these results indicate a significant degree of O₂-limitation of nitrogenase activity in pea and lupin nodules, and that yields may be increased by realizing full potential activity.     

N2 fixation by Acacia species increases under elevated atmospheric CO2

In the present study the effect of elevated CO₂ on growth and nitrogen fixation of seven Australian Acacia species was investigated. Two species from semi‐arid environments in central Australia (Acacia aneura and A. tetragonophylla) and five species from temperate south‐eastern Australia (Acacia irrorata, A. mearnsii, A. dealbata, A. implexa and A. melanoxylon) were grown for up to 148 d in controlled greenhouse conditions at either ambient (350 µmol mol^?1) or elevated (700 µmol mol^?1) CO₂ concentrations. After establishment of nodules, the plants were completely dependent on symbiotic nitrogen fixation. Six out of seven species had greater relative growth rates and lower whole plant nitrogen concentrations under elevated versus normal CO₂. Enhanced growth resulted in an increase in the amount of nitrogen fixed symbiotically for five of the species. In general, this was the consequence of lower whole‐plant nitrogen concentrations, which equate to a larger plant and greater nodule mass for a given amount of nitrogen. Since the average amount of nitrogen fixed per unit nodule mass was unaltered by atmospheric CO₂, more nitrogen could be fixed for a given amount of plant nitrogen. For three of the species, elevated CO₂ increased the rate of nitrogen fixation per unit nodule mass and time, but this was completely offset by a reduction in nodule mass per unit plant mass.     

Grafting experiments on the nature of the decline in N2 fixation during fruit development in soybean

A decline in nitrogen fixation at the time of pod-filling is persistently seen in soybeans. This phenomenon was studied by grafting experiments. Young scions with 3 nodes were grafted near the base of fruiting stocks which had passed their peak of nitrogen fixation. These grafts produced a second peak of nitrogen fixation on the same root system, indicating that the decline is reversible. If the scions were grafted near the apex of the fruiting stocks then the second peak of nitrogen fixation was very small. Thus, the translocation system could have an important role in regulating the decline in nitrogen fixation. Grafting of a second shoot of the same age as the rootstock, after the decline in nitrogen fixation, did not reduce the rate of decline even when the fruits from the scion were removed. It appears that physiological changes in different components of the shoot jointly regulate the decline in the rate of nitrogen fixation in soybeans.     

Gaseous diffusive properties of soybean nodules cultured with non-ambient pO2

Measurements of the short-term response of nodulated roots of soybean ( Glycine max L. Merr, cv. Harosoy: Bradyrhizobium japonicum USDA 16) to rapid changes in surrounding pO₂ indicate that their ability to reversibly adjust gaseous diffusive resistance is retained whether plants are cultured in rhizospheres of very low (2.8%) or very high (61.2%) pO₂. Thus the capacity for reversible short-term diffusion adjustment is additional to structural changes in the fixed diffusional barriers of nodules which allow their continued fixation of N₂ in unfavourably high or low external pO₂. Anatomical evidence, involving quantitative measurement of intercellular spaces in the cortical tissues using electron microscopy of thin sections, indicates that the major fixed diffusional barrier is a boundary layer of cells in the inner cortex which may be as small as one cell thick in nodules from 2.8% O₂ to 5 or 6 cells thick, and almost completely devoid of intercellular spaces, in those from 61.2% O₂. The data are interpreted to indicate that the variable diffusion harrier is distinct from the boundary layer and is most likely to be a property of cells and/or intercellular spaces inside the boundary layer of the nodule cortex.     

Manganese application alleviates the water deficit-induced decline of N2 fixation

Water deficit is a very serious constraint on N₂ fixation rates and grain yield of soybean (Glycine max Merr.). Ureides are transported from the nodules and they accumulate in the leaves during soil drying. This accumulation appears responsible for a feedback mechanism on nitrogen fixation, and it is hypothesized to result from a decreased ureide degradation in the leaf. One enzyme involved in the ureide degradation, allantoate amidohydrolase, is manganese (Mn) dependent. As Mn deficiency can occur in soils where soybean is grown, this deficiency may aggravate soybean sensitivity to water deficit. In situ ureide breakdown was measured by incubating soybean leaves in a 5 mol m ^{? 3} allantoic acid solution for 9 h before sampling leaf discs in which remnant ureide was measured over time. In situ ureide breakdown was dramatically decreased in leaves from plants grown without Mn. At the plant level, allantoic acid application in the nutrient solution of hydroponically grown soybean resulted in a higher accumulation of ureide in leaves and lower acetylene reduction activity (ARA) by plants grown with 0 mol m ^{? 3} Mn than those grown with 6·6 mol m ^{? 3} Mn. Those plants grown with 6·6 mol m ^{? 3} Mn in comparison with those grown with 52·8 mol m ^{? 3} Mn had, in turn, higher accumulated ureide and lower ARA. To determine if Mn level also influenced N₂ fixation sensitivity to water deficit, a dry‐down experiment was carried out by slowly dehydrating plants that were grown in soil under four different Mn nutritions. Plants receiving no Mn had the lowest leaf Mn concentration, 11·9 mg kg ^{? 1}, and had N₂ fixation more sensitive to water deficit than plants treated with Mn in which leaf Mn concentration was in the range of 21–33 mg kg ^{? 1}. The highest Mn treatments increased leaf Mn concentration to 37·5 mg kg ^{? 1} and above but did not delay the decline of ARA with soil drying, although these plants showed a significant increase in ARA under well‐watered conditions.     

Oxygen-induced recovery from short-term nitrate inhibition of N2 fixation in white clover plants from spaced and dense stands

Nitrogenase (N₂ase; EC 1.18.6.1) activity (H₂ evolution) and root respiration (CO₂ evolution) were measured under either N₂:O₂ or Ar:O₂ gas mixtures in intact nodulated roots from white clover ( Trifolium repens L.) plants grown either as spaced or as dense stands. The short-term nitrate (5 m M ) inhibition of N₂-fixation was promoted by competition for light between clover shoots, which reduced CO₂ net assimilation rate. Oxygen-diffusion permeability of the nodule declined during nitrate treatment but after nitrate removal from the liquid medium its recovery parallelled that of nitrogenase activity. Rhizosphere pO₂ was increased from 20 to 80 kPa under N₂:O₂. A simple mono-exponential model, fitted to the nodule permeability response to pO₂, indicated NO⁻₃ induced changes in minimum and maximum nodule O₂-diffusion permeability. Peak H₂ production rates at 80 kPa O₂ and in Ar:O₂ were close to the pre-decline rates at 20 kPa O₂. At the end of the nitrate treatment, this O₂-induced recovery in nitrogenase activity reached 71 and 82%; for clover plants from spaced and dense stands, respectively. The respective roles of oxygen diffusion and phloem supply for the short-term inhibition of nitrogenase activity in nitrate-treated clovers are discussed.     

Effect of Rhizobium sp. BARIRGm901 inoculation on nodulation,nitrogen fixation and yield of soybean (Glycine max) genotypes in gray terrace soil

Soybean plants require high amounts of nitrogen, which are mainly obtained from biological nitrogen fixation. A field experiment was conducted by soybean (Glycine max) genotypes, growing two varieties (Shohag and BARI Soybean6) and two advanced lines (MTD10 and BGM02026) of soybean with or without Rhizobium sp. BARIRGm901 inoculation. Soybean plants of all genotypes inoculated with Rhizobium sp. BARIRGm901 produced greater nodule numbers, nodule weight, shoot and root biomass, and plant height than non-inoculated plants. Similarly, inoculated plants showed enhanced activity of nitrogenase (NA) enzyme, contributing to higher nitrogen fixation and assimilation, compared to non-inoculated soybean plants in both years. Plants inoculated with Rhizobium sp. BARIRGm901 also showed higher pod, stover, and seed yield than non-inoculated plants. Therefore, Rhizobium sp. BARIRGm901 established an effective symbiotic relationship with a range of soybean genotypes and thus increased the nodulation, growth, and yield of soybean grown in gray terrace soils in Bangladesh.     

Growth,nodulation and nitrogen fixation in soybean as affected by air humidity and root temperature

Growth, nodulation and N₂ fixation inGlycine max L. Merr., cv. Biison as affected by the relative humidity of air (RH) during the dark period (95 or 50 – 65 %) and day/night root temperature (T_r) (28/28, 25/25, 18/18, 22/28, 22/18 °C) were studied. The growth parameters (plant fresh and dry mass, yield), nodulation (nodule number and fresh mass) and N₂ fixation abilities (total nitrogen content, nitrogenase activity) increased significantly with the increasing T_r. In addition, at the same T_r during the day all studied parameters were increased at the higher T_r during the dark period. Growth, nodulation and N₂ fixation were significantly enhanced at low RH. The findings indicate that all studied parameters could be regulated by environmental factors during the dark period.     

Salt stress induces a decrease in the oxygen uptake of soybean nodules and in their permeability to oxygen diffusion

The effects of short-term NaCl-salinity on nodules of soybean ( Glycine max L. cv. Kingsoy) were studied on hydroponically-grown plants. Both acetylene reducing activity (ARA) and nodule respiration (O₂ uptake and CO₂ evolution) were immediately inhibited, and the stimulation of them by rising the external partial pressure of O₂ (pO₂) was diminished by the application of 0.1 M NaCl in the nutrient solution. The permeability of the nodule to O₂ diffusion, estimated by O₂ consumption or CO₂ evolution, was significantly lower in the stressed nodules than in the cootrol ones. The respiratory quotient of intact nodules and the ethanol production of excised nodules were increased by low pO₂ and by salt stress. These data confirm that in salt-stressed soybean nodules, O₂ availability is reduced and fermentative pathways are stimulated.     

Decomposition of soybean grown under elevated concentrations of CO2 and O3

A critical global climate change issue is how increasing concentrations of atmospheric CO₂ and ground‐level O₃ will affect agricultural productivity. This includes effects on decomposition of residues left in the field and availability of mineral nutrients to subsequent crops. To address questions about decomposition processes, a 2‐year experiment was conducted to determine the chemistry and decomposition rate of aboveground residues of soybean (Glycine max (L.) Merr.) grown under reciprocal combinations of low and high concentrations of CO₂ and O₃ in open‐top field chambers. The CO₂ treatments were ambient (370 μmol mol^?1) and elevated (714 μmol mol^?1) levels (daytime 12 h averages). Ozone treatments were charcoal‐filtered air (21 nmol mol^?1) and nonfiltered air plus 1.5 times ambient O₃ (74 nmol mol^?1) 12 h day^?1. Elevated CO₂ increased aboveground postharvest residue production by 28–56% while elevated O₃ suppressed it by 15–46%. In combination, inhibitory effects of added O₃ on biomass production were largely negated by elevated CO₂. Plant residue chemistry was generally unaffected by elevated CO₂, except for an increase in leaf residue lignin concentration. Leaf residues from the elevated O₃ treatments had lower concentrations of nonstructural carbohydrates, but higher N, fiber, and lignin levels. Chemical composition of petiole, stem, and pod husk residues was only marginally affected by the elevated gas treatments. Treatment effects on plant biomass production, however, influenced the content of chemical constituents on an areal basis. Elevated CO₂ increased the mass per square meter of nonstructural carbohydrates, phenolics, N, cellulose, and lignin by 24–46%. Elevated O₃ decreased the mass per square meter of these constituents by 30–48%, while elevated CO₂ largely ameliorated the added O₃ effect. Carbon mineralization rates of component residues from the elevated gas treatments were not significantly different from the control. However, N immobilization increased in soils containing petiole and stem residues from the elevated CO₂, O₃, and combined gas treatments. Mass loss of decomposing leaf residue from the added O₃ and combined gas treatments was 48% less than the control treatment after 20 weeks, while differences in decomposition of petiole, stem, and husk residues among treatments were minor. Decreased decomposition of leaf residues was correlated with lower starch and higher lignin levels. However, leaf residues only comprised about 20% of the total residue biomass assayed so treatment effects on mass loss of total aboveground residues were relatively small. The primary influence of elevated atmospheric CO₂ and O₃ concentrations on decomposition processes is apt to arise from effects on residue mass input, which is increased by elevated CO₂ and suppressed by O₃.     

Mechanism of soybean nodule adaptation to different oxygen pressures

Abstract. Soybean nodules showed the ability to adapt to oxygen pressures above and below ambient levels and this adaptation involved a decrease in cortical intercellular air-spaces with increasing oxygen pressure. Nodules were grown in oxygen pressures from 4.7 to 75 kPa and the decrease in number and size of cortical intercellular spaces with increasing oxygen pressure was the result of a change in cell structure and the deposition of an electron dense material within intercellular spaces. Exposure to a saturating pressure of acetylene caused a similar inhibition of respiration and nitrogenase activity in nodules developed in oxygen pressures from 4.7 to 47 kPa, suggesting that putative acetylene-induced changes in oxygen diffusion resistance occur by a different mechanism than that involved in long-term adaptation to oxygen. However, in nodules grown at 75 kPa oxygen, the initial specific activities were lower and did not show an acetylene induced decline. The results are discussed in terms of the current theories of regulation of nitrogenase activity by oxygen availability.     

Modification of NO−3 metabolism in heterocysts of the N2-fixing cyanobacterium Anabaena 7120 (ATCC27893)

Abstract The utilization of NO⁻₃, NO⁻₂ and NH⁺₄ was studied in whole filaments and isolated heterocysts of Anabaena 7120 (ATCC27893). NO⁻₃- and NO⁻₂-uptake were detectable in whole filaments but not in heterocysts, whereas NH⁺₄-uptake was detectable in both. Activity of NO⁻₃-reductase was present in cell-free extracts of whole filaments but not of heterocysts, whereas activities of NO⁻₂-reductase and glutamine synthetase were present in both. NO⁻₃-uptake and reductase activities could not be induced in heterocysts even after prolonged incubation in NO⁻₃ medium. It is suggested that NO⁻₃-metabolism in heterocysts is impaired due to a selective and irreversible loss of NO⁻₃-uptake and reductase systems resulting in the abolition of competition for molybdenum cofactor (Mo-Co) and reductant between nitrogenase and NO⁻₃-reductase, and an increase in glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase levels.     

The relationship between nodule adenylates and the regulation of nitrogenase activity by O2 in soybean

Nodulated soybeans (Glycine max L. Merr, cv. Maple Arrow) were exposed to various physiological and environmental treatments to determine the relationship between nodule adenylate pools and the degree of O₂ limitation of nitrogenase. Adenylate energy charge (AEC = [ATP + 0.5 ADP]/[ATP + ADP + AMP]) and ATP/ADP ratios declined under conditions of decreased (10%) external pO₂ but increased in nodules exposed to elevated (30%) external pO₂. Nitrogenase activity was inhibited by both pO₂ treatments, but recovered towards initial levels within 45 min. AEC also returned to initial levels during this period. To account for these and related data in the literature, it was hypothesized that 1) legume nodules regulate infected cell O₂ concentration (Oi) to maintain adenylate pools at levels which limit respiratory metabolism: 2) treatments which decrease Oi alter the adenylate pools and further limit nodule metabolism; 3) treatments which increase Oi to levels in excess of a narrow range alter the adenylate pools and activate biochemical pathways which are not conducive to nitrogenase activity. In a preliminary test of these hypotheses, changes in AEC and ATP/ADP ratio were studied in nodules in which nitrogenase activity was inhibited by stem girdling, nitrate fertilization and exposure to an Ar:O₂ atmosphere. All three treatments caused an increased O₂ limitation of nodule respiration and nitrogenase activity. However, decreases in AEC were observed only in the stem girdling and nitrate fertilization treatment: Ar:O₂ exposure had no effect on whole nodule AEC. While this result challenged the hypotheses suggesting a central role for adenylates in the regulation of O₂-limited metabolism, it was noted that the Ar:O₂ treatment would differ from the other treatments in that it would have a specific effect on the ATP demands for NH₃ assimilation in the plant fraction. Since AEC and ATP/ADP ratio would be affected by both the rate of ATP synthesis (potentially an O₂-limited process) and the demand for ATP, changes in these parameters in the whole nodule may not be a reliable indicator of adenylate-mediated O₂ limitation. Futher studies are needed to examine in vivo changes in adenylate pools in the plant and bacteroid fractions in nodules which vary in their degree of O₂-limited metabolism.     

Oxygen and the regulation of nitrogen fixation in legume nodules

In N₂-fixing legume nodules, O₂ is required in large amounts for aerobic respiration, yet nitrogenase, the bacterial enzyme that fixes N₂, is O₂ labile. A high rate of O₂ consumptition and a cortical barrier to gas diffusion work together to maintain a low, non-inhibitory O₂ concentration in the central, infected zone of the nodule. At this low O₂ concentration, cytosolic leghemoglobin is required to facilitate the diffusion of O₂ through the infected cell to the bacteria. The resistance of the cortical diffusion barrier is variable and is used by legume nodules to regulate the O₂ concentration in the infected cells such that it limits aerobic respiration and N₂ fixation at all times. The resistance of the diffusion barrier and therefore the degree of O₂ limitation seems to be regulated in response to changes in the O₂ concentration of the central infected zone, the supply of phloem sap to the nodule, and the rate of N assimilation into the end products of fixation.