Effects of drought on nitrogen fixation in soybean root nodules

Soybean plants [Glycine max (L.) Merr.] were grown in silica sand and were drought stressed for a 4 week period during reproductive development and without any mineral N supply in order to maximize demand for fixed nitrogen. A strain of Bradyrhizobium japonicum that forms large quantities of polysaccharide in nodules was used to determine whether or not the supply of reduced carbon to bacteroids limits nitrogenase activity. A depression of 30–40% in nitrogen content in leaves and pods of stressed plants indicated a marked decline in nitrogen fixation activity during the drought period. A 50% increase in the accumulation of bacterial polysaccharide in nodules accompanied this major decrease in nitrogen fixation activity and this result indicates that the negative impact of drought on nodules was not due to a depression of carbon supply to bacteroids. The drought treatment resulted in a statistically significant increase in N concentration in leaves and pods. Because N concentration and chlorophyll concentration in leaves were not depressed, there was no evidence of nitrogen deficiency in drought‐stressed plants, and this result indicates that the negative impact of drought on nodule function was not the cause of the depression of shoot growth. At the end of the drought period, the concentration of carbohydrates, amino nitrogen, and ureides was significantly increased in nodules on drought‐stressed plants. The overall results support the view that, under drought conditions, nitrogen fixation activity in nodules was depressed because demand for fixed N to support growth was lower.     

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

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

Interactions ofRhizobium japonicum strains in soybean nodules and their contribution to nitrogen fixation

Summary Rhizobium japonicum strain 8-0 Str^R applied as inoculum to Clark 63 soybeans formed small ineffective nodules which had very low nitrogenase activity compared to nodules formed by two effective strains, 110 Tet^R and 138 Kan^R. Mean numbers of cells per milligram of nodule tissue for plants up to 34 days old were 7.7×10⁶ for 8-0 Str^R, 4.1×10⁸ for 110 Tet^R and 7.6×10⁸ for 138 Kan^R. Cell counts per unit mass of nodule were independent of plant age for strains 110 Tet^R and 138 Kan^R, however, for strain 8-0 Str^R, 25 and 34 days old plants had fewer viable cells per nodule mass than 18 day old plants. When a mixture of two effective strains was used, the nodules of individual plants were predominantly caused by either 110 Tet^R or 138 Kan^R. In one experiment the predominance was random, but in another, strain 110 Tet^R clearly dominated. Strain 138 Kan^R was absent in some nodules on 18 day old plants, and in others, less than 10² cells per nodule were found. When strains 8-0 Str^R and 138 Kan^R were used as mixed inoculum, most of the nodules had strain 8-0 Str^R but strain 138 Kan^R was detected in many nodules and was generally evident in the largest nodules. Nitrogenase activity by many individual nodules was low except for nodules which had cells of 138 Kan^R. Nitrogenase activity by whole root systems of these plants was relatively high and similar to plants that had only nodules of strain 138 Kan^R. Similar relationships were observed for a mixed inoculum of 8-0 Str^R and 110 Tet^R. In general, mixed inoculations resulted in nodules with a particular strain being dominant for each individual plant. Double infections within individual nodules were not uncommon and such nodules often had disproportionate numbers of cells of two competingR. japonicum strains.Contribution from the Laboratory of Soil Microbiology, Department of Agronomy, Missouri Agricultural Experiment Station. Missouri Journal Series Number 7967.     

A comparison of inhibition of French-bean and soybean nitrogen fixation by nitrate, 1% oxygen or direct assimilate deprivation

Inhibition by NO⁻₃ of acetylene reduction in bean ( Phaseolus vulgaris L. cv. Contender) and soybean ( Glycine max L. cv. Amsoy 71) was measured in parallel with nodule carbohydrate and nitrate metabolism. In bean the onset of inhibition of C₂H₂ reduction (6 h) coincided with decreased import of assimilates and a lowering of carbohydrate pools (sucrose, glucose and starch). Nitrate reductase (EC 1.6.6.1) activity was induced in all plant organs after 3 h but no nitrite was detected in the nodules. In soybean, nodule carbohydrate concentrations and import of assimilates into the nodules increased markedly between 6 to 24 h after supply of nitrate when the nitrogenase (EC 1.7.99.2) was progressively inhibited. High nitrate reductase activity was observed in the nodules and nitrites accumulated because of insufficient nitrite reductase activity. The nitrate-induced inhibition of nitrogenase was compared with the inhibition observed with low oxygen around the roots (1% O²) or with direct assimilate deprivation (girdling or decapitation). Soybean and bean appeared equally sensitive to these treatments as regards to acetylene reduction. The results are discussed in relation to the current hypotheses explaining nitrate-induced inhibition of dinitrogen fixation: assimilate deprivation or nitrite poisoning. Present data are in favour of the first for bean and of the second for soybean.     

Seasonal changes in symbiotic nitrogen fixation and haemoglobin content in nodules of peanuts

Summary Seasonal fluctuations of haemoglobin (Hb) concentration in nodules and the percentage of nitrogen (N) in leaflets of rhizobium-inoculated peanuts from various planting dates, were studied under field conditions.In peanuts planted in the usual season (April, May), no correlation was found between the N and Hb concentrations during the early stages of peanut growth; however, there was a very significant correlation at later periods of plant growth. With July (out-of-season) planting there was no correlation between the Hb and N concentrations at any time.The possibility of evaluating the atmospheric nitrogen (N₂) fixation rate of peanut plants under field conditions by means of Hb and N determinations was studied.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. 1972 Series, no. 2213-E.     

Respiration and oxygen transport in soybean nodules

Summary The respiration rate of individual soybean (Glycine max Merr.) nodules was measured as a function of pO₂ and temperature. At 23°, as the pO₂ was increased from 0.1 to 0.9 atm, there was a linear increase in respiration rate. At 13°, similar results were obtained, except that there was an abrupt saturation of respiration at approximately 0.5 atm pO₂. When measurements were made on the same nodule, the rate of increase in respiration with pO₂ was the same at 13° and 23°. Additional results were that 5% CO in the gas phase had no effect on respiration, except for a small decrease in the pO₂ at which respiration became saturated. Also, nodules still attached to the soybean root displayed the same respiratory behavior as detached nodules. A model for oxygen transport in the nodule is presented which explains these results quantitatively. The essence of the model is that the respiration rate of the central tissue of the nodule is almost entirely determined by the rate of oxygen diffusion to the respiratory enzymes. Evidence is given that the nodule cortex is the site of almost all of the resistance to oxygen diffusion within the nodule.     

Metabolic changes of rhizobia in legume nodules

Bacteria have evolved a wide variety of metabolic strategies to cope with varied environments. Some are specialists and only able to survive in restricted environments; others are generalists and able to cope with diverse environmental conditions. Rhizobia (e.g. Rhizobium, Sinorhizobium, Bradyrhizobium, Mesorhizobium and Azorhizobium species) can survive and compete for nutrients in soil and the plant rhizosphere but can also form a beneficial symbiosis with legumes in a highly specialized plant cell environment. Inside the legume-root nodule, the bacteria (bacteroids) reduce dinitrogen to ammonium, which is secreted to the plant in exchange for a carbon and energy source. A new and challenging aspect of nodule physiology is that nitrogen fixation requires the cycling of amino acids between the bacteroid and plant. This review aims to summarize the metabolic plasticity of rhizobia and the importance of amino acid cycling.     

Role of oxygen limitation and nitrate metabolism in the nitrate inhibition of nitrogen fixation by pea

The impact of nitrate (5–15 m M , 2 to 7 days) on nitrogenase activity and nodule-oxygen limitation was investigated in nodulated, 21-day-old plants of a near-isogenic nitrate reductase-deficient pea mutant (A3171) and its wild-type parent ( Pisum sativum L. cv. Juneau). Within 2 days, 10 or 15 m M nitrate, but not 5 m M nitrate, inhibited the apparent nitrogenase activity (measured as in situ hydrogen evolution from nodules of intact plants) of wild-type plants; none of these nitrate levels inhibited the apparent nitrogenase activity of A3171 plants. Nodule-oxygen limitation, measured as the ratio of total nitrogenase activity to potential nitrogenase activity, was increased in both wild-type and A3171 plants by all nitrate treatments. By 3 to 4 days the apparent nitrogenase activity of A3171 and wild-type plants supplied with 5 m M nitrate declined to 53 to 69% of control plants not receiving nitrate. By 6 to 7 days the apparent nitrogenase activity of A3171 plants was similar to the control value whereas that of the wild-type plants continued to decline. From 3 to 7 days, no significant differences in nodule-oxygen limitation were observed between the nitrate (5 m M ) and control treatments. The results are interpreted as evidence for separate mechanisms in the initial (O₂ limitation) and longer-term (nitrate metabolism) effects of nitrate on nitrogen fixation by effectively nodulated pea.     

Dark chilling inhibition of photosynthesis and symbiotic nitrogen fixation in soybean during pod filling

The growth stage of a soybean [Glycine max (L.) Merrill] plant may influence its physiological response to dark chilling. Opposed to vegetative development, the intense nutrient and energy requirements of the developing seeds during pod filling could cause additional chilling damage and decreased recovery capacity. Previously, we investigated dark chilling tolerance during vegetative development in two soybean genotypes, 'Maple Arrow' and 'Fiskeby V' and consistently found that photosynthesis and symbiotic nitrogen fixation (SNF) was less affected by dark chilling in 'Maple Arrow'. In this study we describe the dark chilling response of the same genotypes during pod filling. Our aim was to establish whether the potential selection criteria for dark chilling tolerance, identified during vegetative development, was equally sensitive during pod filling. The results indicate that photosynthesis is less affected by dark chilling in 'Maple Arrow' than in 'Fiskeby V', not only during vegetative development, but also during the critical reproductive stage of pod filling. 'Fiskeby V' also lacks the ability to restore normal photosynthetic capacity during an extended recovery treatment. The decrease of nodule ureide content indicates that SNF was inhibited to a similar extent in both genotypes. Nodule ureide content was reduced more than stem ureide content, suggesting that the former is a more sensitive indicator of chilling stress effects on SNF. The results indicate that certain photosynthetic and fluorescence parameters are sensitive indicators of dark chilling tolerance throughout plant development and should prove valuable in future breeding programmes aimed at increasing the chilling tolerance of soybean.     

Regulation of respiration and the oxygen diffusion barrier in soybean protect symbiotic nitrogen fixation from chilling-induced inhibition and shoots from premature senescence

Symbiotic nitrogen fixation is sensitive to dark chilling (7 degrees C-15 degrees C)-induced inhibition in soybean (Glycine max). To characterize the mechanisms that cause the stress-induced loss of nodule function, we examined nodule structure, carbon-nitrogen interactions, and respiration in two soybean genotypes that differ in chilling sensitivity: PAN809 (PAN), which is chilling sensitive, and Highveld Top (HT), which is more chilling resistant. Nodule numbers were unaffected by dark chilling, as was the abundance of the nitrogenase and leghemoglobin proteins. However, dark chilling decreased nodule respiration rates, nitrogenase activities, and NifH and NifK mRNAs and increased nodule starch, sucrose, and glucose in both genotypes. Ureide and fructose contents decreased only in PAN nodules. While the chilling-induced decreases in nodule respiration persisted in PAN even after return to optimal temperatures, respiration started to recover in HT by the end of the chilling period. The area of the intercellular spaces in the nodule cortex and infected zone was greatly decreased in HT after three nights of chilling, an acclimatory response that was absent from PAN. These data show that HT nodules are able to regulate both respiration and the area of the intercellular spaces during chilling and in this way control the oxygen diffusion barrier, which is a key component of the nodule stress response. We conclude that chilling-induced loss of symbiotic nitrogen fixation in PAN is caused by the inhibition of respiration coupled to the failure to regulate the oxygen diffusion barrier effectively. The resultant limitations on nitrogen availability contribute to the greater chilling-induced inhibition of photosynthesis in PAN than in HT.     

Formation of nodules on non-nodulating soybean T201 after treatment with 2,4-dichlorophenoxyacetate

The formation of effective root nodules on a non-nodulating line (T201) of soybean (Glycine max (L.) Merr.,) was induced by a treatment with 2,4-dichlorophenoxyacetate (2,4-D). The induced nodules, inoculated with mixed Bradyrhizobium japonicum strains A1017 and IRj2101, had a normal internal structure, red in colour and the cells being filled with bacteroids. Externally, the induced nodules were of unusual shape, being paired or gourd-like in form and were attached to thickened roots. The nodules were capable of acetylene reduction (3.1–3.5 moles g^-1 fresh weight nodules h^-1), allowing the growth of plants with dark green leaves.     

Potential for increasing biological nitrogen fixation in soybean

The importance of soybean as a source of oil and protein, and its ability to grow symbiotically on low-N soils, point to its continued status as the most valuable grain legume in the world. With limited new land on which to expand, and emphasis on sustainable systems, increases in soybean production will come mostly from increased yield per unit area. Improvements in biological nitrogen fixation can help achieve increased soybean production, and this chapter discusses research and production strategies for such improvement.The soybean-Bradyrhizobium symbiosis can fix about 300 kg N ha^-1 under good conditions. The factors which control the amount of N fixed include available soil N, genetic determinants of compatibility in both symbiotic partners and lack of other yield-limiting factors. Response to inoculation is controlled by the level of indigenous, competing bradyrhizobia, the N demand and yield potential of the host, and N availability in the soil.Research efforts to improve BNF are being applied to both microbe and soybean. While selection continues for effective, naturally occurring bradyrhizobia for inoculants and the use of improved inoculation techniques, genetic research on bradyrhizobia to improve effectiveness and competitiveness is advancing. Selection, mutagenesis and breeding of the host have focused on supernodulation, restricted nodulation of indigenous B. japonicum, and promiscuous nodulation with strains of bradyrhizobia from the cowpea cross-inoculation group. The research from the host side appears closer to being ready for practical use in the field.Existing knowledge and technology still has much to offer in improving biological nitrogen fixation in soybean. The use of high-quality inoculants, and education about their benefits and use can still make a significant contribution in many countries. The importance of using the best adapted soybean genotype with a fully compatible inoculant cannot be overlooked, and we need to address other crop management factors which influence yield potential and N demand, indirectly influencing nitrogen fixation. The implementation of proven approaches for improving nitrogen fixation in existing soybean production demands equal attention as received by research endeavours to make future improvements.     

Effect of plant genotype and nitrogen fertilizer on symbiotic nitrogen fixation by soybean cultivars

Summary Isotopic as well as non-isotopic methods were used to assess symbiotic nitrogen fixation within eight soybean [Glycine max (L.) Merr.] cultivars grown at 20 and 100 kg N/ha levels of nitrogen fertilizer under field conditions.The¹⁵N methodology revealed large differences between soybean cultivars in their abilities to support nitrogen fixation. In almost all cases, the application of 100 kg N/ha resulted in lower N₂ fixed in soybean than at 20 kg N/ha in the first year of the study. However, N₂ fixed in one cultivar, Dunadja, was not significantly affected by the higher rate of N fertilizer application. These results were confirmed by measurements of acetylene reduction activity, nodule dry weight and N₂ fixed as measured by the difference method. Further proof of differences in N₂ fixed within soybean cultivars and the ability of Dunadja to fix similar amounts of N₂ at 20 and 100 kg N/ha was obtained during a second year experiment. Dunadja yield was affected by N fertilizer and produced larger yield at 100 kg N/ha than at 20 kg N/ha. This type of cultivar could be particularly useful in situations where soil N levels are high or where there is need to apply high amounts of N fertilizer.The present study reveals the great variability between legume germplasms in the ability to fix N₂ at different inorganic N levels, and also the potential that exists in breeding for nitrogen fixation associative traits. The¹⁵N methodology offers a unique tool to evaluate germplasms directly in the field for their N₂ fixation abilities at different N fertilizer levels.