Regulation of soybean nitrogen fixation in response to rhizosphere oxygen: I. Role of nodule respiration

Nitrogen fixation (acetylene reduction) rates of nodules on intact field-grown soybean (Glycine max) subjected to altered oxygen concentration (0.06-0.4 cubic millimeter per cubic millimeter) returned to initial rates during an 8-hour transitory period. Hydroponically grown soybean plants also displayed a transitory (1-4 hours) response to changes in the rhizosphere oxygen concentration after which the fixation rates returned to those observed under ambient oxygen concentrations. It was hypothesized that soybean nodules contain a regulatory mechanism which maintains a stable oxygen concentration inside nodules at a sufficiently low concentration to allow nitrogenase to function. A possible physiological mechanism which could account for this regulation is adjustment in nodule respiration activity such that nodule oxygen concentration and nitrogen fixation are maintained at stable levels. Experiments designed to characterize the non-steady-state oxygen response and to test for the presence of nodule respiratory control are presented. Non-steady-state acetylene reduction and nodule respiration (oxygen uptake) rates measured after alterations in the external oxygen concentration indicated that the regulatory mechanism required 1 to 4 hours to completely adjust to changes in the external oxygen concentration. Steady-state nodule respiration, however, did not respond to alterations in the rhizosphere oxygen concentration. It was concluded that soybean nodules can adjust to a wide range of rhizosphere oxygen concentrations, but the mechanism which controls nitrogen fixation rates does not involve changes in the nodule respiration rate.     

The occurrence of haemoglobin and hydrogenase in nodules of twelve Casuarina-Frankia symbiotic associations

Seedlings of Casuarina cunninghamiana Miq., C. equisetifolia L. and C. glauca Sieber growing in N-deficient potting mix were inoculated with Frankia sp. from each of 4 different sources. After ca 4 months, plants were harvested and nodules from the 12 Casuarina-Frankia combinations evaluated for (1) concentrations of haemoglobin (measured as CO-reactive haem) and (2) occurrence of hydrogenase. The aim was to determine if these factors were related to nitrogen accumulation and biomass production. There were marked host-Frankia source interactions with up to 10-fold differences in plant dry weight and 50-fold differences in the efficiency of nitrogen fixation (as estimated by N₂ accumulated mg^?1 nodule dry weight). Differences in plant growth and nitrogen accumulation were apparently related to nodule specific activity, because the 12 associations had similar nodulation characteristics, e.g. time for nodulation to occur. The concentration of haemoglobin in Casuarina nodules ranged from 0 to 27 nmol haem (g FW)^?1. There was a strong linear correlation between concentrations of haemoglobin and dry weights of the whole plants (r=0.77, 0.92 and 0.97, P≤0.05) for C. cunninghamiana, C. equisetifolia and C. glauca symbiotic associations, respectively. However, the linear correlation between concentration of haemoglobin and nitrogen content of whole plant was lower (r=0.60, 0.64 and 0.71, P≤0.05) for the three Casuarina symbioses, respectively, and there was only a poor correlation between haemoglobin concentration in nodules and the rate of nitrogen accumulation on nodule weight basis. This indicates that haemoglobin concentration is not the sole physiological determinant of nitrogen fixation in Casuarina. All the Casuarina-Frankia symbiotic associations studied also showed the presence of a hydrogen uptake enzyme. The activity of the enzyme ranged from 5.1 to 34.1 μmol H₂ (g FW)^?1 h^?1, and hydrogen uptake was not correlated with plant dry weight, nitrogen content or the rate of nitrogen fixation. Hydrogen evolution could not be detected in any of the associations.     

Effects of drought stress on legume symbiotic nitrogen fixation: physiological mechanisms

Drought stress is one of the major factors affecting nitrogen fixation by legume-rhizobium symbiosis. Several mechanisms have been previously reported to be involved in the physiological response of symbiotic nitrogen fixation to drought stress, i.e. carbon shortage and nodule carbon metabolism, oxygen limitation, and feedback regulation by the accumulation of N fixation products. The carbon shortage hypothesis was previously investigated by studying the combined effects of CO2 enrichment and water deficits on nodulation and N2 fixation in soybean. Under drought, in a genotype with drought tolerant N2 fixation, approximately four times the amount of 14C was allocated to nodules compared to a drought sensitive genotype. It was found that an important effect of CO2 enrichment of soybean under drought was an enhancement of photo assimilation, an increased partitioning of carbon to nodules, whose main effect was to sustain nodule growth, which helped sustain N2 rates under soil water deficits. The interaction of nodule permeability to O2 and drought stress with N2 fixation was examined in soybean nodules and led to the overall conclusion that O2 limitation seems to be involved only in the initial stages of water deficit stresses in decreasing nodule activity. The involvement of ureides in the drought response of N2 fixation was initially suspected by an increased ureide concentration in shoots and nodules under drought leading to a negative feedback response between ureides and nodule activity. Direct evidence for inhibition of nitrogenase activity by its products, ureides and amides, supported this hypothesis. The overall conclusion was that all three physiological mechanisms are important in understanding the regulation of N2 fixation and its response of to soil drying.     

Effects of calcium deficiency on symbiotic nitrogen fixation

The mechanism of the effect of mild calcium deficiency on nitrogen metabolism of the symbiotic plant was studied from the distribution of calcium and of nitrogen and carbohydrate fractions in plant organs.

Nitrogen concentrations of all plant organs decreased with calcium deficiency. Addition of either a nitrogen or a calcium salt increased nitrogen concentrations. For roots as well as whole plants the effects of one salt were largely replaced by the other. These effects establish that calcium deficiency decreased the supply of fixed nitrogen from nodules to other organs. As weight of nodules was independent of calcium it follows that nodular efficiency was impaired.

Since nitrogen concentrations of nodules decreased rather than increased with calcium deficiency it is suggested that calcium deficiency interferes with the rate of reduction of nitrogen in the nodule rather than with the export of reduced nitrogen. Distribution of α-amino, amide, and ammonium nitrogen are consistent with this suggestion.

Nitrogen fixation was not limited by translocation of carbohydrates to nodules as calcium deficiency had little effect on the concentration of soluble carbohydrates and actually increased the concentration of starch in nodules.

Calcium deficiency depressed the calcium content of nodules so that nitrogen fixation may have been impaired by inadequate calcium for nodular structure or metabolism.

     

野生大豆（Glycine soja）酰脲含量与根瘤固氮活力的关系

野生大豆（Ｇｌｙｃｉｎｅｓｏｊａ）酰脲含量与根瘤固氮活力的关系朱长甫,苗以农,刘学军,许守民（东北师范大学生命科学学院,长春１３００２４）郑惠玉,徐豹（吉林省农业科学院大豆研究所,公主岭１３６１００）关键词：野生大豆,固氮活力,酰脲,蛋白质根据固氮豆...     

Regulation of Soybean Nitrogen Fixation in Response to Rhizosphere Oxygen: II. Quantification of Nodule Gas Permeability

Nodule nitrogen fixation rates are regulated by a mechanism which is responsive to the rhizosphere oxygen concentration. In some legumes, this oxygen-sensitive mechanism appears to involve changes in the gas permeability of a diffusion barrier in the nodule cortex. In soybean evidence for such a mechanism has not been found. The purpose of this research was to make quantitative measurements of soybean nodule gas permeability to test the hypothesis that soybean nodule gas permeability is under physiological control and responsive to the rhizosphere oxygen concentration. Intact hydroponically grown soybean plants were exposed to altered rhizosphere oxygen concentrations, and the nodule gas permeability, acetylene reduction and nodule respiration rates were repeatedly assayed. After a change in the external oxygen concentration, nitrogenase activity and nodule respiration rates displayed a short-term transient response after which the values returned to rates similar to those observed under ambient oxygen conditions. In contrast to steady-state nitrogenase activity and nodule respiration, nodule gas permeability was dramatically affected by the change in oxygen concentration. Decreasing the external oxygen concentration to 0.1 cubic millimeter per cubic millimeter resulted in a mean increase in nodule gas permeability of 63%. Increasing the rhizosphere oxygen concentration resulted in decreased nodule gas permeability. These data are consistent with the hypothesis that soybean nodules are capable of regulating nitrogen fixation and nodule respiration rates in response to changes in the rhizosphere oxygen concentration and indicate that the regulatory mechanism involves physiological control of the nodule gas permeability.     

Growth and root nodule nitrogenase activity of Pisum sativum as influenced by transpiration

The influence of shoot transpiration on the rates of growth and nitrogen fixation was investigated in Pisum sativum L. cv. Rondo. In short term experiments, rates of transpiration and acetylene reduction of intact plants were measured simultaneously, using air-tight perspex vessels enclosing the basal part of the nodulated root. In long term experiments, accumulation of dry matter and reduced nitrogen in the plant were determined as well. Transpiration rate changed diurnally and was varied by manipulating the vapour saturation deficit or the flow rate of the air in the growth cabinet. The rate of acetylene reduction declined after subjecting intact plants to high transpiration rates. This decline was accompanied by a desiccation of the root nodules. Dry matter and reduced nitrogen accumulation were not affected by transpiration rate. At low transpiration rate reduced nitrogen content of the root nodules was higher than at high transpiration rate. However, in these nodules the rate of acetylene reduction was not significantly affected. It is concluded that the nitrogenase activity of pea root nodules is insensitive to changes in the flow rate and the organic N concentration of the xylem sap within a wide range of transpiration conditions under the applied growth conditions.     

Review article. Symbiotic N2 fixation response to drought

Symbiotic nitrogen fixation is highly sensitive to drought, which results in decreased N accumulation and yield of legume crops. The effects of drought stress on N2 fixation usually have been perceived as a consequence of straightforward physiological responses acting on nitrogenase activity and involving exclusively one of three mechanisms: carbon shortage, oxygen limitation, or feedback regulation by nitrogen accumulation. The sensitivity of the nodule water economy to the volumetric flow rate of the phloem into the nodule offers a common framework to understand each of these mechanism. As these processes are sensitive to volumetric phloem flow into the nodules, variations in phloem flow as a result of changes in turgor pressure in the leaves are likely to cause rapid changes in nodule activity. This could explain the special sensitivity of N2 fixation to drying soils. It seems likely that N feedback may be especially important in explaining the response mechanism in nodules. A number of studies have indicated that a nitrogenous signal(s), associated with N accumulation in the shoot and nodule, exists in legume plants so that N2 fixation is inhibited early in soil drying. The existence of genetic variation in N2 fixation response to water deficits among legume cultivars opens the possibility for enhancing N2 fixation tolerance to drought through selection and breeding.     

土壤氮磷添加下豆科草本植物生物固氮与磷获取策略的权衡机制

豆科草本植物固氮是陆地生态系统重要的自然氮输入方式, 影响着草地生产的经济性和可持续性。为探讨氮磷交互作用影响豆科草本植物生物固氮率的潜在生理生态机制, 该研究选取8种豆科草本植物分别种植在对照、氮肥添加、磷肥添加和氮磷耦合添加处理的土壤中, 进行野外盆栽实验。测定了初花期植物生物量和营养含量、根部碳水化合物含量、根际pH、根际柠檬酸含量、根际有效磷含量、植物根瘤生物量、磷含量及其生物固氮率。主要结果: 依赖于豆科物种, 氮添加显著促进了豆科草本植物根际磷的活化, 降低了根生物量分配以及根系非结构性碳水化合物含量。在两种磷添加处理下, 氮添加导致8种豆科草本植物根瘤生物量平均下降27%-36%, 生物固氮率平均下降20%-33%。磷添加降低了根际的磷活化, 但促进了豆科草本植物根系发育和非结构性碳水化合物的积累。在施氮和不施氮条件下, 磷添加分别使8种豆科草本植物的生物固氮率提高了45%-69%和0-47%。氮添加降低豆科草本植物生物固氮率, 其原因是氮添加提高了植物磷需求, 为活化更多磷, 豆科草本植物降低根系生物量和根系非结构性碳水化合物的含量, 导致根瘤发育受到限制。在氮添加的同时进行磷添加, 能够改善土壤氮磷平衡, 促进根系生长和非结构性碳水化合物积累, 缓解了增氮对生物固氮的抑制作用。     

Soybean genotypic differences in sensitivity of symbiotic nitrogen fixation to soil dehydration

Nitrogen fixation activity by soybean (Glycine max (L.) Merr.) nodules has been shown to be especially sensitive to soil dehydration. Specifically, nitrogen fixation rates have been found to decrease in response to soil dehydration preceding alterations in plant gas exchange rates. The objective of this research was to investigate possible genetic variation in the sensitivity of soybean cultivars for nitrogen fixation rates in response to soil drying. Field tests showed substantial variation among cultivars with Jackson and CNS showing the least sensitivity in nitrogen accumulation to soil drying. Glasshouse experiments confirmed a large divergence among cultivars in the nitrogen fixation response to drought. Nitrogen fixation in Jackson was again found to be tolerant of soil drying, but the other five cultivars tested, including CNS, were found to be intolerant. Experiments with CNS which induced localized soil drying around the nodules did not result in decreases in nitrogen fixation rates, but rather nitrogen fixation responded to drying of the entire rooting volume. The osmotic potential of nodules was found to decrease markedly upon soil drying. However, the decrease in nodule osmotic potential occurred after significant decreases in nitrogen fixation rates had already been observed. Overall, the results of this study indicate that important genetic variations for sensitivity of nitrogen fixation to soil drying exist in soybean, and that the variation may be useful in physiology and breeding studies.     

Nitrogen fixation and plant growth of common bean (Phaseolus vulgaris L.) at different levels of phosphorus availability

The efficacy of the alumina system for differentiating between bean (Phaseolus vulgaris L.) genotypes for growth at different levels of phosphorus availability was determinated. In addition to response to P levels, comparisons were made between plants receiving N either from fertilizer or nitrogen fixation. When the cv. Carioca was provided with either 100 ppm of N or inoculated withRhizobium leguminosarum biovarphaseoli, differences in shoot dry weight and nodule number were related to P level. There was a greater proportion of green, ineffectivevs. red, active nodules at the low P concentration than at the higher P concentration. In a second experiment, two cvs., Puebla 152 and Carioca and the breeding line UW 24-21, either were inoculated with rhizobia or provided with 150ppm of N. Each genotype-nitrogen combination was grown at 8 levels of P. There was a positive effect of P level on shoot dry weight, nodule number and nodule mass. Root mass was affected less than nodule or shoot mass by the P level of the growth medium. Nodule mass, but not P concentration in the nodules, was affected by P level, whereas in the other plant tissues, P concentrations were lower at lower P levels in the media.     

Variability in the response of six genotypes of N<Subscript>2</Subscript>-fixing <Emphasis Type="Italic">Medicago ciliaris</Emphasis> to NaCl

Genotypic variability was assessed within six Medicago ciliaris genotypes growing symbiotically with Sinorhizobium medicae in order to identify physiological criteria (growth, ion content, and plant health) associated with salt tolerance. Response to salt stress depended on the line and the level of salt. Two lines with lower dry biomass under non-saline conditions (TNC 1.8 from a semi-arid area and TNC 10.8 from a sub-humid area), were more tolerant to NaCl, whereas the most productive lines (TNC 11.5 and TNC 11.9 from a humid bioclime) were more sensitive in terms of growth and nitrogen fixation. Susceptibility of symbiotic nitrogen fixation to saline stress was not associated with a higher accumulation of Na⁺ in nodules, since the most tolerant lines TNC 1.8 and TNC 10.8 accumulated the highest Na⁺ amount in nodules. Leaf area and net photosynthate assimilation rate were conserved in line TNC 1.8 and to a lesser extent in line TNC 10.8 potentially owing to a greater ability to protect aerial organs and nodules from Na⁺ damage and to insure a better supply of leaves with nitrogen. Our results suggest that nodule growth and number and nodule Na⁺ content should not be used as selection tools for tolerance or susceptibility, since two of the tested lines maintained consistent growth in spite of reduced nodule and high Na⁺ content. Instead, the most reliable physiological indicators for tolerance appear to be consistent growth (i.e., no growth changes) and reduced leaf Na⁺ accumulation with increasing concentrations of NaCl.     

Influence of zinc on cadmium-induced toxicity in nodules of pigeonpea (Cajanus cajan L. Millsp.) inoculated with arbuscular mycorrhizal (AM) fungi

Arbuscular mycorrhizal (AM) fungi are known to alleviate heavy-metal stress in plants. The intent of the present work was to analyze accumulation of heavy metals (Cd and Zn) in nodules of two Cajanus cajan (L.) Millsp. genotypes and their subsequent impact on nitrogen fixation, oxidative stress, and non-protein thiols (glutathione and phytochelatins) with and without AM fungus Glomus mosseae. Accumulation of Cd and Zn in nodules resulted in sharp reduction in nodule number, nodule dry mass as well as nitrogen fixation (leghemoglobin and nitrogenase (N₂ase)), although Cd had more pronounced effects than Zn. Cd-induced lipid peroxidation, H₂O₂ accumulation, and electrolyte leakage were largely reversed by Zn supplementation. Zn application significantly altered the negative effects of Cd on the synthesis of non-protein thiols, suggesting antagonistic behaviour of Zn. Higher concentration of Zn was more effective in lessening the negative effects of Cd than its lower concentration. Remarkable genotypic variation was found, with more severe effects of both the metals in P792 than Sel 85N. Glomus mosseae attenuated the phytotoxic effects of metals in nodules by decreasing metal uptake, oxidative stress, and by enhancing defense system ultimately leading to better nitrogen-fixing potential of pigeonpea nodules.     

The physiology and biochemistry of cultivar-strain interactions in the white clover-Rhizobium symbiosis

Two white clover cultivars were inoculated with two Rhizobium leguminosarum bv. trifolii strains in a factorial series of experiments. Plants were grown in axenic conditions in nitrogen free nutrient solution in a controlled environment room. Variations in nitrogen fixation were dependent partly upon general strain effects, partly upon general cultivar effects but there were also substantial differences attributable to precise interactions between specific combinations. The physiological and biochemical basis of these differences was examined. There were variations in the onset of nodulation and nitrogenase (acetylene reduction) activity. The rate at which nitrogenase activity developed also differed between associations as did the average size and number of nodules but none of these effects correlated well with differences in plant dry matter accumulation. Studies on nodule biochemistry revealed that the major nitrogen fixation enzymes were present in all four associations. Nodule protein content and enzyme activity (on a g nodule fresh weight basis) were substantially greater in associations formed by the more effective strain but cannot explain the interactive effect on dry matter accumulation. The relevance of these data to our understanding of factors regulating variations in nitrogen fixation is discussed.     

大豆-根瘤菌共生体系光合与固氮关系研究

水培大豆和田间生长的大豆,接种根瘤菌 Rhizobium B16-11C 后植株全氮含量、叶片叶绿素含量和净光合速率及种子产量都明显增加。比较 Clark 大豆的结瘤品系和不结瘤品系获类似结果。摘除根瘤后3天内叶片净光合速率无明显变化。大豆植株遮阴、去叶或切掉地上部导致根瘤活性明显下降。但去豆荚不能提高根瘤固氮的比活性。根瘤活性的日变化不能用根瘤蔗糖、淀粉含量或周围温度的变化来解释,其控制因子尚待深入研究。     

Studies on the Relationship Between Photosynthesis and Nitrogen Fixation in the Symbiotic System of Soybean and Nodule Bacteria( Rhizobium)

The relationship between photosynthesis of soybean and nitrogen fixation of the nodules by symbiotic Rhizobium was studied. The contents of total nitrogen and chlorophyll, the net photosynthetic rate and seed yield of soybean were much higher in either hydroponically cultivated or field-grown plants inoculated with Rhizobium B16–11C (or Clark nodulating strain) than in control without inoculation (or Clark non-nodulating strain). These results show that the symbiotic nitrogen fixation has a beneficial effect on photosynthesis. However, the effect was indirect and slow so that there was no change in the net photosynthetic rate of the soybean leaves until three clays after removing nodules from the soybean roots. On the other hand, decreasing the photosynthate supply to nodule by shade, defoliation or shoot removal of the soybean, the nodule activity declined significantly. It seems that the supply of photosynthate to root nodule is a limiting factor for symbiotic nitrogen fixation. However, the diurnal variation of the nodule activity could not be explained by change neither in the contents of sucrose and starch of the root nodules nor in the ambient temperature. The factor controlling the diurnal variation deserves further study.     

Growth, nitrogen fixation and ammonium assimilation in common bean (Phaseolus vulgaris L) subjected to iron deficiency

A greenhouse experiment was carried out aiming to study the effect of iron deficiency on nitrogen fixation and ammonium assimilation in common bean nodules. Host-plant and nodule growth, symbiotic nitrogen fixation, glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were analyzed in two common bean varieties subjected to iron deficiency. Results showed that host-plant and nodules growth, nitrogen fixation and GS activity decreased when under Fe-deficiency against an important increase of ammonium accumulation and GDH activity. Tolerant variety Flamingo is clearly less affected by iron deficiency than the sensitive one, Coco blanc. The allocation of iron to nodules and Fe use-efficiency for nodule growth and symbiotic nitrogen fixation were on the basis of the symbiotic performance of Flamingo under iron deprivation. Under Fe-deficiency, GDH take over GS the ammonium assimilation activity, particularly in the tolerant variety.     

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.     

Inhibition of Nitrogen Fixation in Soybean Plants Supplied with Nitrate I. Nitrite Accumulation and Formation of Nitrosylleghemoglobin in Nodules

The accumulation of nitrite in nodules was investigated to elucidatethe mechanism of inhibition of nitrogen fixation in nodulesof soybean (Glycine max. [L.] Merr.) plants supplied with nitrate.Acetylene-reducing activity (ARA) in nodules fell within 24h as a result of the supply of exogenous nitrate, accompaniedby an increase in the accumulation of nitrite in the cytosolbut not in the bacteroids of nodules. Nitrate reductase (NR)activity in the nodule cytosol remained high, irrespective ofthe supply of nitrate. Nitrosylleghemoglobin (LbNO) was detectedspectrophotometrically in the extract from nodules in whichnitrogen fixation was inhibited by nitrate. In experiments invitro, it was found that LbNO was easily formed from leghemoglobinin the presence of nitrite and dithionite. Thus, it is suggested that nitrogen fixation was inhibited primarilyby a decrease in the function of leghemoglobin, attributableto the formation of LbNO, which was caused by the accumulationof nitrite generated from nitrate by NR in the nodule cytosol. (Received August 22, 1989; Accepted January 24, 1990)