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J. Kevin Vessey 《Plant and Soil》1994,158(2):151-162
The closed acetylene reduction assay has been used as a measure of nitrogenase activity and an indicator of N2 fixation in Rhizobium/legume symbioses for 25 years. However, starting 10 years ago this assay has come under harsh criticism as being inaccurate. Currently, confusion exists regarding the conditions under which the acetylene reduction assay can be used accurately, or whether it can be used at all as a measure of nitrogenase activity. This article reviews the circumstance that has lead to this confusion. The author argues that under the proper assay conditions and with the appropriate checks, the closed acetylene reduction assay is still a valuable tool in assessing relative differences in nitrogenase activity in Rhizobium/legume symbioses. 相似文献
3.
Mathematical modeling of oxygen diffusion and respiration in legume root nodules 总被引:8,自引:2,他引:6
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Denison RF 《Plant physiology》1992,98(3):901-907
The O2 permeability of legume root nodules is under physiological control; decreases in permeability are triggered by various forms of stress. Two linked mathematical models were used to explore several hypotheses concerning the physical nature of the variable diffusion barrier in nodules. Respiration and diffusion of dissolved O2 and oxygenated leghemoglobin were simulated for the nodule cortex and the nodule interior. Measured nodule permeabilities were shown to be inconsistent with the hypothesis that large numbers of air-filled pores penetrate the diffusion barrier. Changes in the affinity of leghemoglobin for O2 or in the rate of cytoplasmic streaming in diffusion barrier cells did not result in the large changes in O2 permeability reported for real nodules. The presence or absence, but not the thickness, of aqueous plugs in radial pores through the cortex was found to have a large effect on permeability. Flooding of intercellular spaces, either between layers of cells in the cortex or in the nodule interior, also caused large changes in simulated permeability. The unsteady-state O2 method for determining nodule permeability was tested using data generated by the model. The accuracy of the method was confirmed, provided that certain assumptions (full oxygenation of leghemoglobin under pure O2 and uniform conditions in the nodule interior) are met. 相似文献
4.
Catalase activity and nitrogen fixation in legume root nodules 总被引:1,自引:0,他引:1
5.
After 40 days of growth at 25°C, Lotus pedunculatus cav., cv. Maku plants infected with Rhizobium loti strain NZP2037 displayed similar relative growth rates but had twice the nodule mass and only one third the whole plant dry weight of plants infected with Bradyrhizobium sp. (Lotus) strain CC814s. In the NZP2037 symbiosis, the rate of CO2 evolution (per g dry weight of nodulated root) was 1.6 times as high as that in the CC814s symbiosis while the rate of C2H2 reduction (per g dry weight of nodule) was only 48% of that in the CC814s symbiosis. Studies of the effect of short term temperature changes on the gas exchange characteristics (CO2 and H2 evolution, C2H2 reduction) of these symbioses revealed wide differences in the optima for C2H2 reduction. Nodules infected with NZP2037 displayed maximal C2H2 reduction rates [157 μmol (g dry weight nodule)?1 h?1] at 12°C, whereas nodules infected with CC814s were optimal at 30°C [208 μmol (g dry weight nodule)?1 h?1]. These short term studies suggested that differences in temperature optima for N2 may have partially accounted for the poorer effectivity, at 25°C, of strain NZP2037 when compared with strain CC-814s. The relative efficiency [RE = 1 – (H2 evolution/C2H2 reduction)] of N2 fixation varied widely with temperature in the two symbioses, but there was a general trend toward higher RE with lower temperatures. The ratio of CO2 evolution: C2H2 reduction (mol/mol) in nodulated roots infected with CC814s was constant (ca 10 CO2/C2H2) between 5°C and 30°C, whereas in plants infected with NZP2037 it reached a minimal value of 3.3 CO2/C2H2 at 10°C and was 19 CO2/C2H2 at the growing temperature (25°C). 相似文献
6.
Variation in nitrogenase and hydrogenase activity of alaska pea root nodules 总被引:5,自引:6,他引:5
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Hydrogenase activity of root nodules in the symbiotic association between Pisum sativum L. and Rhizobium leguminosarum was determined by incubating unexcised nodules with tritiated H2 and measuring tissue HTO. Hydrogenase activity saturated at 0.50 millimolar H2 and was not inhibited by the presence of 0.10 atmosphere C2H2, which prevented H2 evolution from nitrogenase. Total H2 production from nitogenase was estimated as net H2 evolution in air plus H2 exchange in 0.10 atmosphere C2H2. Although such an estimate of nitrogenase function may not be quantitatively exact, due to uncertain relationships between H2 exchange and H2 uptake activity of hydrogenase, differences observed in H2 exchange under various conditions represent an indication of changes in hydrogenase activity. Hydrogenase activity was lower in associations grown under higher photosynthetic photon flux densities and decreased relative to total H2 production by nitrogenase. Total H2 production and hydrogenase activity were maximum 28 days after planting. Thereafter, hydrogenase activity and H2 production declined, but the potential proportion of nitrogenase-produced H2 recovered by the uptake hydrogenase system increased. Of five R. leguminosarum strains tested two possessed hydrogenase activity. Strains which had the potential to reassimilate H2 had significantly higher rates of N2 reduction than those which did not exhibit hydrogenase activity. 相似文献
7.
Effects of carbohydrate on the internal oxygen concentration, oxygen uptake, and nitrogenase activity in detached pea nodules 总被引:2,自引:2,他引:0
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The interaction between carbon substrates and O2 and their effects on nitrogenase activity (C2H2) were examined in detached nodules of pea (Pisum sativum L. cv “Sparkle”). The internal O2 concentration was estimated from the fractional oxygenation of leghemoglobin measured by reflectance spectroscopy. Lowering the endogenous carbohydrate content of nodules by excising the shoots 16 hours before nodule harvest or by incubating detached nodules at 100 kPa O2 for 2 hours resulted in a 2- to 10-fold increase in internal O2, and a decline in nitrogenase activity. Conversely, when detached nodules were supplied with 100 millimolar succinate, the internal O2 was lowered. Nitrogenase activity was stimulated by succinate but only at high external O2. Oxygen uptake increased linearly with external O2 but was affected only slightly by the carbon treatments. The apparent diffusion resistance in the nodule cortex was similar in all of the treatments. Carbon substrates can thus affect nitrogenase activity indirectly by affecting the O2 concentration within detached nodules. 相似文献
8.
The kinetics of asymbiotic nitrogenase activity in three strains of the actinomycete Frankia were studied. Decay rates for enzyme activity were determined by adding chloramphenicol to active acetylene-reducing cells and measuring the time required for all activity to cease. Synthesis rates were measured by bubbling oxygen through actively-reducing cells (which totally destroyed all activity) and then measuring the time required for activity to return to normal. Decay rates (t
1/2) for these three strains were approximately 30 to 40 min. Synthesis rates were slower and initial nitrogenase activities were recorded about 110 min (DDB 011610) or 210 min (DDB 020210 and WgCc1.17) after return to air-equilibrated cultures. Frankia strain WgCc1.17 showed a greater sensitivity to oxygen and nitrogenase activity was totally lost when cells were bubbled only with atmospheric concentrations of oxygen. The results presented here indicate that nitrogenase activity turnover time is relatively rapid, on the order of minutes rather than hours or days. However, regulation of nitrogenase activity will differ from one strain to another and asmmbiotic characterization will be useful for understanding nitrogenase regulation in the bacterial-plant symbiosis.Contribution no. 879 from the Battelle-Kettering Laboratory 相似文献
9.
The dramatic decrease in nitrogenase activity after the defoliation of forage legumes has been recognized for a long time; however, the underlying mechanisms are not understood yet. The impact of current photosynthesis can be excluded. The precise role of carbohydrate availability is still unclear and remains to be established. From current knowledge we can conclude that, after defoliation, nitrogenase activity in legume nodules is down-regulated by a variable oxygen diffusion resistance. The triggering elements are not known; there is, however, increasing evidence that the plant's demand for symbiotically fixed nitrogen plays an important role. The possibility is here discussed that, after defoliation, a nitrogen feedback mechanism regulates nitrogenase activity through a variable oxygen diffusion resistance in the nodules. 相似文献
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11.
Dakora FD 《Annals of botany》1995,75(1):49-54
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. 相似文献
12.
The rate of C2H2 reduction by nodulated seedlings of Elaeagnus angustifolia (Russian olive) was followed as a function of time. Our goals were to: 1) determine whether there is an C2H2-induced decline in nitrogenase activity; and 2) investigate the mechanism of any decline. We found a peak rate of C2H2 reduction at 1.5 min after the introduction of C2H2 that was followed by a rapid decline in activity to 56% of the peak value. After the decline there was a partial recovery to 67% of the peak value at 60 min. When the pO2 was decreased during the decline there was no significant effect (p0.05) on nitrogenase activity. When the C2H2 reduction assay was preceded by an incubation in a gas mixture (20 kPa O2) with Ar substituted for N2, there was little decline in nitrogenase activity as a function of time, but the rate of C2H2 reduction per gram nodule was reduced by approximately 50%. From these results we conclude that t Elaeagnus angustifolia exhibits a pronounced C2H2-induced decline and consequently the initial peak rate C2H2 reduction must be determined to obtain a valid measure of nitrogenase activity. We further suggest that cessation of NH3 formation initiates the decline and that the decline is not caused by a change in nodule permeability to gases. 相似文献
13.
Stephen Rawsthorne Frank R. Minchin Rodney J. Summerfield Claire Cookson James Coombs 《Phytochemistry》1980,19(3):341-355
The literature concerning the metabolism of carbon compounds during the reduction, assimilation and translocation of nitrogen in root nodules of leguminous plants is reviewed. The reduction of dinitrogen requires an energy source (ATP) and a reluctant which are both supplied by respiratory catabolism of carbohydrates produced by the host plant. Photosynthates are also required to generate the carbon skeletons for amino acid or urcide synthesis during the assimilation of ammonia produced by the bacteria within the nodule tissue. Competition for photosynthates occurs between the bacteroids, nodule tissue and the various vegetative and reproductive sinks in the host plant. The nature of carbon compounds involved in these processes, their routes of metabolism, the mechanisms of control and the partitioning of metabolises between the various sites of utilization are only poorly understood. It is apparent that dinitrogen is reduced to ammonia in the bacteroids. Both fast- and slow-growing strains of Rhizobium possess the Entner-Doudoroff pathway of glucose catabolism, and some, if not all, enzymes of the Emden-Meyerhof pathway. Some bacterial cultures also metabolize carbon through the ketogluconate pathway but only the fast-growing strains of cultured rhizobia possess the key enzyme of the pentose phosphate pathway (6-phosphogluconate dehydrogenase). The host cells are thought to contain the complete Emden-Meyerhof pathway and tricarboxylic acid cycle, which provides the carbon skeletons for assimilation of the ammonia, formed by the bacteroids, into α-amino acids. A pathway of anapleurotic carbon conservation, operative in the host cells, synthesizes oxaloacetic acid through β-carboxylation of phosphoenol pyruvate. This process could be important in the recapture and assimilation of respired CO2 in the rhizosphere. The main route of assimilation of ammonia produced by the bacteroids would appear to be via the glutamine synthetase-glutamate synthase pathway in the host cells. However, glutamate dehydrogenase may also be involved in ammonia assimilation. These enzymes also occur in in vitro cultures of Rhizobium and in bacteroids where they presumably participate in the synthesis of amino acids for growth of the bacteria or bacteroids. Nitrogen assimilated into glutamine or glutamate is exported from the nodules in a variety of forms, which include asparagine, glutamine, aspartate, homoserine and allantoates, in proportions which depend on the legume species. Studies on regulation of the overall process have focussed on expression of bacteroid genes and on the control of enzyme activity, at the level of nitrogenase and enzymes of nitrogen assimilation in particular. However, due to the wide range of experimental techniques, environmental conditions and plant species which have been used, no clear conclusions can yet be drawn. The pathways of carbon flow in nitrogen metabolism, particularly in relation to the synthesis of ureides and the regulation of carbon metabolism, remain key areas for future research in symbiotic nitrogen fixation. 相似文献
14.
High-performance liquid chromatography (HPLC) with fluorescence detection was used to study thiol metabolism in legume nodules. Glutathione (GSH) was the major non-protein thiol in all indeterminate nodules examined, as well as in the determinate nodules of cowpea (Vigna unguiculata), whereas homoglutathione (hGSH) predominated in soybean (Glycine max), bean (Phaseolus vulgaris), and mungbean (Vigna radiata) nodules. All nodules had greater thiol concentrations than the leaves and roots of the same plants because of active thiol synthesis in nodule tissue. The correlation between thiol tripeptides and the activities of glutathione synthetase (GSHS) and homoglutathione synthetase (hGSHS) in the nodules of eight legumes, and the contrasting thiol contents and activities in alfalfa (Medicago sativa) leaves (98% hGSH, 100% hGSHS) and nodules (72% GSH, 80% GSHS) indicated that the distribution of GSH and hGSH is determined by specific synthetases. Thiol contents and synthesis decreased with both natural and induced nodule senescence, and were also reduced in the senescent zone of indeterminate nodules. Thiols and GSHS were especially abundant in the meristematic and infected zones of pea (Pisum sativum) nodules. Thiols and gamma-glutamylcysteinyl synthetase were also more abundant in the infected zone of bean nodules, but hGSHS was predominant in the cortex. Isolation of full-length cDNA sequences coding for gamma-glutamylcysteinyl synthetase from legume nodules revealed that they are highly homologous to those from other higher plants. 相似文献
15.
Reactive oxygen species and antioxidants in legume nodules 总被引:35,自引:0,他引:35
Manuel Becana David A. Dalton Jose F. Moran Iñaki Iturbe-Ormaetxe Manuel A. Matamoros Maria C. Rubio 《Physiologia plantarum》2000,109(4):372-381
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. 相似文献
16.
T Ruiz-Argüeso D W Emerich H J Evans 《Biochemical and biophysical research communications》1979,86(2):259-264
Some strains of rhizobia possess a hydrogenase system which catalyzes the oxidation of the H2 that is evolved from nitrogenase during N2 fixation. Oxidation of H2 by a hydrogen uptake positive strain of provides energy for support of the N2 fixation reactions and protects nitrogenase from O2 damage 相似文献
17.
Matamoros MA Loscos J Coronado MJ Ramos J Sato S Testillano PS Tabata S Becana M 《Plant physiology》2006,141(3):1068-1077
Ascorbic acid (vitamin C) is a major antioxidant and redox buffer, but is also involved in other critical processes of plants. Recently, the hypothesis has been proposed that legume nodules are unable to synthesize ascorbate and have to import it from the shoot or root, thus providing a means by which the plant regulates nodule senescence. The last step of ascorbate biosynthesis in plants is catalyzed by L-galactono-1,4-lactone dehydrogenase (GalLDH). The mRNAs encoding GalLDH and three other enzymes involved in ascorbate biosynthesis are clearly detectable in nodules. Furthermore, an active membrane-bound GalLDH enzyme is present in nodule mitochondria. Biochemical assays on dissected nodules reveal that GalLDH activity and ascorbate are correlated in nodule tissues and predominantly localized in the infected zone, with lower levels of both parameters (relative to the infected tissues) in the apex (87%) and senescent region (43%) of indeterminate nodules and in the peripheral tissues (65%) of determinate nodules. In situ RNA hybridization showed that the GalLDH mRNA is particularly abundant in the infected zone of indeterminate and determinate nodules. Thus, our results refute the hypothesis that ascorbate is not synthesized in nodules and lend support to a previous conclusion that ascorbate in the infected zone is primarily involved in the protection of host cells against peroxide damage. Likewise, the high ascorbate and GalLDH activity levels found in the apex of indeterminate nodules strongly suggest a participation of ascorbate in additional functions during symbiosis, possibly related to cell growth and division and to molecular signaling. 相似文献
18.
Nitrogenase (EC 1.7.99.2) activity of a cowpea (Vigna unguiculata (L.) Walp cv Caloona) symbiosis formed with a Rhizobium strain (176A27) lacking uptake hydrogenase and maintained under conditions of a 12-hour day at an air temperature of 30°C (800-1000 microeinsteins per square meter per second) and a 12-hour night at an air temperature of 20°C showed a marked diurnal variation in ratio of nitrogen fixed to hydrogen evolved. As little as 0.3 micromole nitrogen was fixed per micromole hydrogen evolved in the photoperiod versus up to 0.6 in the dark period. In plants maintained under the same diurnal illumination regime but at constant (day and night) air temperature (30°C), this difference was abolished and a relatively constant ratio of nitrogen fixed to hydrogen evolved (around 0.3 micromole per micromole) was observed day and night. Exposure of nodulated roots to a range of temperatures maintained for 2 hours in a single photoperiod indicated that, whereas hydrogen evolution increased with increasing temperature from 15°C to a maximum around 35°C, nitrogen fixation was largely unaffected over this temperature range. Both functions of the enzyme declined sharply at temperatures above 38°C. A similar general response of nitrogen fixation to root temperature was observed in glasshouse-grown, sand-cultured plants maintained under a range of temperatures (from 15 to 35°C) for a 14-day period in mid vegetative growth. The effect of temperature on the proportion of electrons allocated to proton reduction compared with nitrogen reduction showed a linearly increasing relationship (correlation coefficient = 0.96) between 15°C and 47°C. 相似文献
19.
In search of the mechanism of nitrate inhibition of nitrogenase activity in legume nodules: Recent developments 总被引:4,自引:0,他引:4
The mechanisms involved in the inhibition of nitrogenase activity in legume nodules by nitrate is unclear. This paper reviews and evaluates proposed mechanisms of this inhibition. Emphasis is placed on recent developments, which suggest that nitrate causes an O2 limitation of nitrogenase activity. Several mechanisms that involve a nitrate-induced increase in resistance to O3 diffusion in the nodule cortex are discussed. 相似文献
20.
The possibility that respiration limits oxygen access to nitrogenase was tested by artificially upsetting the balance between oxygen consumption (respiration) and oxygen influx (diffusion). Argon treatment of the nodulated root system on intact plants stopped in vivo nitrogenase activity almost completely. Upon return to air, nitrogenase activity was very low and recovered gradually to full activity after about 5 h. In vitro measurements on nodule homogenates indicated that active nitrogenase was lost upon the shift from low (argon) to normal (air) oxygen. Maintenance of nodulated root systems at low temperature (2°C) inhibited both respiration and in vivo nitrogenase activity. Upon return to normal temperature (22°C), oxygen uptake recovered very rapidly, but nitrogenase activity recovered only gradually to full activity after about 5 to 6 h. Again, loss of active nitrogenase could, at least partly, explain the reduced in vivo nitrogenase activity. The effects from a temporarily impaired balance between oxygen consumption and oxygen influx thus point to the importance of respiration for limiting oxygen access to nitrogenase. 相似文献