Growth and nitrogen fixation in Lotus japonicus and Medicago truncatula under NaCl stress: nodule carbon metabolism

Lotus japonicus and Medicago truncatula model legumes, which form determined and indeterminate nodules, respectively, provide a convenient system to study plant-Rhizobium interaction and to establish differences between the two types of nodules under salt stress conditions. We examined the effects of 25 and 50mM NaCl doses on growth and nitrogen fixation parameters, as well as carbohydrate content and carbon metabolism of M. truncatula and L. japonicus nodules. The leghemoglobin (Lb) content and nitrogen fixation rate (NFR) were approximately 10.0 and 2.0 times higher, respectively, in nodules of L. japonicus when compared with M. truncatula. Plant growth parameters and nitrogenase activity decreased with NaCl treatments in both legumes. Sucrose was the predominant sugar quantified in nodules of both legumes, showing a decrease in concentration in response to salt stress. The content of trehalose was low (less than 2.5% of total soluble sugars (TSS)) to act as an osmolyte in nodules, despite its concentration being increased under saline conditions. Nodule enzyme activities of trehalose-6-phosphate synthase (TPS) and trehalase (TRE) decreased with salinity. L. japonicus nodule carbon metabolism proved to be less sensitive to salinity than in M. truncatula, as enzymatic activities responsible for the carbon supply to the bacteroids to fuel nitrogen fixation, such as sucrose synthase (SS), alkaline invertase (AI), malate dehydrogenase (MDH) and phosphoenolpyruvate carboxylase (PEPC), were less affected by salt than the corresponding activities in barrel medics. However, nitrogenase activity was only inhibited by salinity in L. japonicus nodules.     

A comparative developmental pattern of enzymes of carbon metabolism and pentose phosphate pathway in mungbean and lentil nodules

The activities of enzymes of pentose phosphate pathway (PPP) viz. glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and carbon metabolism viz. phosphoenol pyruvate carboxylase, NADP- isocitrate dehydrogenase and NADP-malic enzyme were measured in the plant and bacteroid fractions of mungbean (ureide exporter) and lentil (amide exporter) nodules along with the developing roots for comparison. The enzymes of pentose phosphate pathway in legume cytosol had higher activities at a stage of maximum nitrogenase activity and higher sucrose metabolism. However, bacteroids had only limited capacity for this pathway. The specific activities of these enzymes were greater in ureide than in amide exporter. CO₂ fixation via higher activity of phosphoenolpyruvate carboxylase in the plant part of the nodules in lentil might have been due to the greater synthesis of four carbon amino acids for amide export. The peak of NADP-isocitrate dehydrogenase in both legumes coincided with the pentose phosphate pathway enzymes at the time of high rates of sucrose metabolism and nitrogen fixation. Higher activities of NADP-malic enzyme were obtained in mungbean than in the lentil nodules. These findings are consistent with the role of these enzymes in providing reductant (NADPH) and substrates for energy yielding metabolism of bacteroids and carbon skeletons for ammonia assimilation.     

Effects of salt stress on growth, photosynthesis and nitrogen fixation in chick-pea (Cicer arietinum L.)

Plants of chick-pea (Cicer arietinum L. cv. ILC1919) inoculated with Mesorhizobium ciceri strain ch-191 were grown in a controlled environmental chamber, and were administered salt (0, 50, 75, and 100 mM NaCl) during the vegetative period. Four harvests (4, 7, 11, and 14d after treatment) were analysed. The aim was to ascertain whether the negative effect of saline stress on nitrogen fixation is due to a limitation on the photosynthate supply to the nodule or a limitation on the nodular metabolism which sustains nitrogenase activity.Plant growth was affected only by the highest NaCl concentration, whereas nitrogenase activity was affected from 50 mM. At the first harvest, Rubisco, PEPC and MDH activities in leaves rose with salt, but fell during the following harvests. The increase of PEPC and MDH in nodules at the two first samplings was clearly related to salt concentration. While 50 mM NaCl increased GS and GOGAT in nodules at some harvests, 100 mM strongly inhibited these activities at all the harvests. The accumulation of proline, amino acids and carbohydrates was clearly related to salt especially in the leaves, whereas in the nodules the protein content was boosted by salt. Although photosynthesis declined with NaCl, the response of nitrogen fixation to salt was more pronounced. This situation, together with carbohydrate accumulation, suggests that the lack of photosynthate does not cause the inhibition of nitrogenase activity under this type of stress. The similar trend observed for the PEPC-MDH pathway and the ARA support the hypothesis concerning the limitation in the supply of energy substrate, mainly malate, to the bacteroids. The accumulation of compatible solutes is more a consequence of damage produced by salt stress than of a protective strategy.     

La Dendrologia di Ulisse Aldrovandi

Abstract

Legume-Rhizobium symbiotic nitrogen (N₂) fixation plays a critical role in sustainable nitrogen management in agriculture. The nitrogen fixed by the root nodules not only affects the nitrogen cycle of nature, but is also of great economic importance. A number of physiological and biochemical processes in the nodules are affected by salt stress. The objective of this study was to evaluate the role of arbuscular mycorrhiza (AM) in moderating toxic effects of salt stress on nodular metabolism in Cajanus cajan (L.) Millspaugh (pigeonpea) cv. Manak. Exposure of plants to salinity stress (4, 6 and 8 dSm^?1) caused ionic imbalance, which resulted in increased Na⁺ and reduced K⁺ and Ca²⁺ contents in the nodules. Salinity induced increased synthesis and accumulation of proline and glycine betaine. Salt stress significantly increased the antioxidant enzyme activities in the nodules of all plants. Nodular growth suffered remarkably and a marked decline in nodule biomass was observed under salt stress. Leghemoglobin content and acetylene reduction activity (ARA) also declined under saline conditions. AM could significantly improve nodule dry mass, leghemoglobin content and nitrogenase activity, and phosphorus content under salt stress. Activities of antioxidant enzymes increased markedly in nodules of mycorrhizal-stressed plants. This study suggested a correlation between improved functional efficiency of nodules and higher osmolyte accumulation and enhanced antioxidant enzyme activities of AM plants under stressed conditions relative to the nodules of uninoculated plants.     

Carbon metabolism and bacteroid respiration in nodules of chick-pea (Cicer arietinum L.) plants grown under saline conditions

ABSTRACT

The present work investigates the relationships between nitrogen fixation, carbon metabolism and oxygen consumption by bacteroids of Mesorhizobium ciceri in root nodules of chick-pea plants. Its aim was to establish whether some of the compounds which accumulate under salt stress may be used as respiratory substrates by bacteroids to fuel their own metabolism and nitrogenase activity. Plants were grown in a growth chamber, and salt stress was induced by adding 50 mM NaCl to the nutrient solution at sowing. The data presented here show a rise in fermentative metabolism in nodules of chick-pea plants exposed to high salinity, and suggest that proline, lactate or ethanol, may play an important role as energy-yielding substrates for bacteroids in this plant species. The bacteroids could utilize glucose as a respiratory substrate both under control and saline conditions, while malate did not appear to be the preferred substrate in the presence of salt.     

Comparative study of nitrogen fixation and carbon metabolism in two chick-pea (Cicer arietinum L.) cultivars under salt stress

Two cultivars of Cicer arietinum with differential tolerance to salinity have been compared by analysing growth, photosynthesis, nodulation, nitrogenase activity, and carbon metabolism in the nodule cytosol. The aim was to help elucidate the relationships between, on the one hand, sucrose and malate metabolism in nodules and, on the other, the inhibition of nitrogen fixation under salt stress. Chick-pea cultivars Pedrosillano (sensitive) and ILC1919 (tolerant) inoculated with Mesorhizobium ciceri strain Ch-191 were grown in a controlled environmental chamber and were treated with salt (0, 50, 75, and 100 mM NaCl) from sowing to harvest time (28 d). Plant growth and photosynthesis were more affected by salt in Pedrosillano than in ILC1919. Also the effect of salt on nodulation and nitrogen fixation was much more pronounced in Pedrosillano. The increase in nodular mass in ILC1919 can partially counteract the inhibition of nitrogenase activity. The enzymes of sucrose breakdown were inhibited by NaCl, but in ILC1919 a rise in alkaline invertase was observed with salinity, which could compensate for the lack of the sucrose synthase hydrolytic activity. The activity of PEPC was stimulated by salt in ILC1919. Also, this cultivar showed higher malate concentrations in root nodules.     

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

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

Water-deficit effects on carbon and nitrogen metabolism of pea nodules

Two experiments were carried out to investigate the effects of water-deficit stress on carbon and nitrogen metabolism of Pisum sativum nodules. In the first experiment, leaf w was allowed to reach -1.0 MPa over a period of 14 d whilst in the second experiment -1.5 MPa was reached during the same time period. Nodule activities of phosphoenol pyruvate carboxylase, glutamine synthetase, alkaline invertase, pyruvate decarboxylase, alcohol dehdyrogenase, uridine pyro-phosphorylase, and malate dehydrogenase activities were not affected by water-deficit stress. In the first experiment (-1.0 MPa), sucrose synthase (SS), an enzyme which hydrolyses sucrose to support nodule metabolism, declined by 50% in activity and about 25% in content, according to Western immunoblot data. In the second experiment (-1.5 MPa), SS activity decreased by 75% together with glutamate synthase and aspartate aminotransferase which declined by 60% and 40%, respectively. Coincident with the decline of these activities, a dramatic increase in the nodule content of sucrose and a slight increase in the levels of total free amino acids were found. It has been recently suggested that the decline in SS activity and, therefore, a reduced potential to metabolize sucrose may be an important factor contributing to the overall response of soybean nodules to water stress. These results suggest that this observation may be also correct for temperate legumes with indeterminate nodules. However, in this latter case, the activity of some enzymes involved in nitrogen assimilation (glutamate synthase and aspartate aminotransferase) were also affected by water-deficit stress).Key words: Pisum sativum, water stress, nitrogen metabolism, nodule metabolism, pea, sucrose synthase.      

The regulation of nodulation,nitrogen fixation and ammonium assimilation under a carbohydrate shortage stress in the Discaria trinervis-Frankia symbiosis

N₂-fixation is sensitive to limitation in the availability of newly synthesised carbohydrates for the nodules. We decided to explore the response of the D. trinervis - Frankia symbiosis to a transient decrease in carbohydrate supply to nodules. Feedback inhibition of nodulation as well as nodule growth was not released by a 6-day dark stress in D. trinervis nodulated plants. However, nitrogen fixation and assimilation were affected by the imposed stress. Nitrogenase activity was totally inhibited after 4 days of darkness although high levels of nitrogenase components were still detected at this time. Degradation of FeMo and Fe nitrogenase subunits – both at similar rates – was observed after 6 days of dark stress, revealing the need for inactivation to precede enhancement of protein turnover. Glutamine synthetase (GS), malate dehydrogenase (MDH) and asparagine synthetase (AS) polypeptides were also degraded during the dark stress, although at a lower rate than nitrogenase. ARA and nitrogenase were totally recovered 8 days after resuming normal illumination. It seems that current nitrogenase activity and ammonium assimilation are not, or are only weakly linked with the feedback control of nodulation in D. trinervis. These observations give support to the persistence of an autoregulatory signal in mature nodules that is not sensitive to transient shortages of carbon supply and sustains the inhibition of nodulation in the transient absence of N₂ fixation.     

Effect of hydro- and osmopriming of chickpea (Cicer arietinum L.) seeds on enzymes of sucrose and nitrogen metabolism in nodules

Three year data on the effect of water- and mannitol (4%) priming of chickpea seeds (12 h at 25°C) showed higher number and biomass of nodules in the plants from primed seeds than from non-primed seeds. The biomass of nodules increased to 75 DAS but decreased by 90 DAS. Activities of sucrose metabolism enzymes (sucrose synthase (SS) and alkaline invertase) and of nitrogen metabolism (glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH)) in nodules of primed and non-primed crops during development are reported. SS and alkaline invertase activities increased to 70 DAS and then decreased. In primed plants, the higher SS activity in nodules at 60 and 70 DAS might be responsible for providing more energy and carbon skeleton for nitrogen fixation and for ammonium assimilation in primed plants. At 85 DAS, though the SS activity decreased in comparison with the earlier growth stages, it was still higher in nodules of the primed crops than the non-primed crop. Activity of alkaline invertase was maximum at 70 DAS in the nodules of primed and non-primed crops. Priming increased nodule GS activity at 70 and 85 DAS. GOGAT activity was unaffected by priming but GDH activity was greater in nodules from primed crops at 50 DAS. Elevated SS and GS nodule activities in primed chickpeas might be responsible in increasing nodule biomass and metabolic activity thereby increasing seed fill.     

Comparative Proteomic Analysis of Soybean Nodulation Using a Supernodulation Mutant,SS2-2

Legumes have the ability to form root nodules that fix atmospheric nitrogen through a symbiotic interaction with nitrogen-fixing bacteria. As a first step in dissecting the molecular process of nodulation, proteome reference maps of soybean roots and nodules were constructed. Time course analysis revealed that the transition from root to nodule was accompanied with downregulation of defense-response related proteins, including Mn-superoxide dismutase, peroxidase (Prx), PR10, and stress-induced protein, leading to the initiation of a symbiotic interaction between the two partners. Following nitrogenase biosynthesis, the host plant cooperated with the rhizobia to fix atmospheric nitrogen under microaerobic conditions via expression of leghemoglobins and antioxidant proteins. Comparative proteome analysis indicated lower expression of malate dehydrogenase (MDH), leghemoglobins and nitrogenase in the nodule development of the supernodulation mutant, SS2-2, as compared to the wild type, indicating that SS2-2 forms functionally immature nodules in higher numbers with the lower activity of nitrogen fixation.     

The role of sucrose synthase in the response of soybean nodules to drought

Experiments were carried out to investigate the effects of droughtstress on enzymatic activities related to carbon and nitrogenmetabolism in soybean nodules. Gradual drought stress, imposedby withholding water nutrients, resulted in declines in thewater potential of leaves and nodules consistent with a significantdecline in N2 fixation. However, the amounts of nitrogenasecomponents 1 and 2 were virtually unaffected by drought stress.Similarly, no significant changes could be detected in aspartateaminotransferase, phosphoenolpyruvate carboxylase, glutaminesynthetase or alkaline invertase activities throughout the experiment.In contrast, sucrose synthase (SS), one of the enzymes involvedin sucrose metabolism in legume nodules, declined dramaticallyin activity and in content within a few days of withholdingwater. Coincident with this decline in SS activity were significantincreases in the nodule contents of sucrose, total free aminoacids and ureides. The amounts of proline, however, did notincrease until some days later. It is suggested that SS mayplay a key role in the regulation of nodule carbon metabolismand, therefore, of nitrogen fixation under drought stress conditions. Key words: Glycine max, soybean, nodule metabolism, drought stress, sucrose synthase     

The role of formate metabolism in nitrogen fixation in Rhizobium Spp.

Formate metabolism supported nitrogen-fixation activity in free-living cultures of Rhizobium japonicum. However, formate0dependent nitrogense activity was observed only in the presence of carbon sources such as glutamate, ribose or aspartate which by themselves were unable to support nitrogenase activity. Formate-dependent nitrogenase activity was not detected in the presence of carbon sources such as malate, gluconate or glycerol which by themselves supported nitrogenase activity. A mutant strain of R. japonicum was isolated that was unable to utilise formate and was shown to lack formate dehydrogenase activity. This mutant strain exhibited no formate-dependent nitrogenase activity. Both the wild-type and mutant strains nodulated soybean plants effectively and there were no significant differences in the plant dry weight or total nitrogen content of the respective plants. Furthermore pea bacteroids lacked formate dehydrogenase activity and exogenously added formate had no stimulatory effect on the endogenous oxygen uptake rate. The role of formate metabolism in symbiotic nitrogen fixation is discussed.Abbreviation FDH formate dehydrogenase     

Alleviation of Salt Stress in Common Bean (Phaseolus vulgaris) by Exogenous Abscisic Acid Supply

In this work the effect of abscisic acid (ABA) and 100 mM NaCl on common bean (Phaseolus vulgaris var. Coco) growth, nitrogenase activity, and nodule metabolism was studied. Experiments were carried out in a controlled environmental chamber and plants, at the vegetative growth stage (16 days old), were treated with ABA (1 μM and 10 μM) and 48 h later were exposed to saline treatment. Results revealed that plant dry weight, nodule dry weight, nitrogen fixation (acetylene reduction activity and ureides content), and most enzymes of ammonium and ureides metabolism were affected by both ABA and NaCl. The addition of 1 μM ABA to the nutrient solution before the exposure to salt stress reduced the negative effect of NaCl. Based on our results, we suggest that ABA application improves the response of Phaseolus vulgaris symbiosis under saline stress conditions, including the nitrogen fixation process and enzymes of ammonium assimilation and purine catabolism.     

Development of the nitrogen fixing apparatus in the legumes,Centrosema pubescens Benth., and Vigna unguiculata L. Walp.

The sequence of events leading up to the establishment of symbiotic nitrogen-fixation were studied in two tropical legumes, Centrosema pubescens Benth, and Vigna unguiculata L. Walp. Parameters measured included fresh and dry weights, chlorophyll and leghaemoglobin contents, as well as the activities of NADH-nitrate reductase (EC 1.6.6.1), and nitrogenase (nitric-oxide reductase-EC 1.7.99.2) in plants that were inoculated with suitable rhizobia or which were watered with potassium nitrate. Dry weight and photosynthetic activity of both species followed the sigmoidal pattern which is characteristic of most plants. Growth was little different in either a qualitative or quantitative sense whether nitrogen was supplied as nitrate or through dinitrogen fixation. Although the biochemical sequence of events was dependent on the limiting sensitivities of the individual assays used, the data suggest that nitrate reductase is the first measurable enzymatic activity in the nodules (and roots), followed by acetylene reduction and leghaemoglobin in that order. It is possible therefore, that low levels of symbiotic nitrogen fixation occur in the nodules in the absence of leghaemoglobin. Nitrate reductase activity in C. pubescens nodules was negatively exponentially correlated with nitrogenase activity of the same nodules, suggesting a changing metabolism in old nodules. These data are discussed in terms of environmental and physical factors known to control nitrogen fixation.     

Ontogenetic interactions between photosynthesis and symbiotic nitrogen fixation in pigeonpea

Total nodule nitrogenase activity (TNA, μmols ethylene plant^-1 h^-1) in pigeonpea (Cajanus cajari) increased with plant growth to reach maximum at flowering (75 days after sowing), decreasing thereafter until maturity (120 days after sowing). However, specific nodule nitrogenase activity (SNA, μmols ethylene g^-1 nodule fresh wt h^-1) reached its maximum earlier (45 days after sowing). The rate of photosynthesis and shoot and nodule respiration followed a similar pattern to TNA. However, higest rates of root respiration were observed at flowering and again immediately before final harvest. ¹⁴CO₂ feeding studies showed that assimilates produced in leaves before flowering were retained in the vegetative parts. Assimilates produced after flowering were exported to the reproductive structure at the expense of the nodules. It is suggested that the decreased availability of photosynthate to nodules decreased nitrogen fixation.     

Nitrogen distribution index ofCajanus cajan L. during drought and rehydration

Relative competition among various plant parts for N during water stress,i.e. nitrogen distribution index (NDI) was determined in relation to specific nitrogenase activity (SNA) and nodule and soil nitrogen in both indeterminate (H-77-216) and determinate (ICPL-151) types of pigeonpea (Cajanus cajan L.) under greenhouse conditions. Two levels of water stress,i.e. moderate (soil Ψ_w) -0.77 MPa) and severe (soilΨ_w -1.34 MPa) were created by witholding the irrigation at vegetative (40 DAS) and flowering (70 DAS) stages. At vegetative stage under moderate stress the highest NDI was in nodules of cv. H-77-216 and in leaf of cv. ICPL-151. Under severe stress both the cultivars showed negative values of NDI, with maximum loss of N from root and nodules. Cultivar ICPL-151 behaved differently at flowering and vegetative stages. Very high loss of N from different plant parts was seen at flowering under severe stress. All the plant parts showed gain in N during rehydration. Loss and gain in N at both the stages under stress and rehydration respectively, correlated with available N in soil. Specific nitrogenase activity (SNA) and nodule N were maximum at moderate stress and related with NDI values of leaf and nodules.     

Sucrose Synthase in Legume Nodules Is Essential for Nitrogen Fixation

The role of sucrose synthase (SS) in the fixation of N was examined in the rug4 mutant of pea (Pisum sativum L.) plants in which SS activity was severely reduced. When dependent on nodules for their N supply, the mutant plants were not viable and appeared to be incapable of effective N fixation, although nodule formation was essentially normal. In fact, N and C resources invested in nodules were much greater in mutant plants than in the wild-type (WT) plants. Low SS activity in nodules (present at only 10% of WT levels) resulted in lower amounts of total soluble protein and leghemoglobin and lower activities of several enzymes compared with WT nodules. Alkaline invertase activity was not increased to compensate for reduced SS activity. Leghemoglobin was present at less than 20% of WT values, so O₂ flux may have been compromised. The two components of nitrogenase were present at normal levels in mutant nodules. However, only a trace of nitrogenase activity was detected in intact plants and none was found in isolated bacteroids. The results are discussed in relation to the role of SS in the provision of C substrates for N fixation and in the development of functional nodules.     

Nitrogen fixation by Anabaena cylindrica

Summary Blending Anabaena cylindrica cultures results in a loss of nitrogenase activity which is correlated with the breakage of the filaments at the junctions between heterocysts and vegetative cells. Oxygen inhibition of nitrogen fixation was significant only above atmospheric concentrations. Nitrogen-fixation activities in the dark were up to 50% of those observed in the light and were dependent on oxygen (10 to 20% was optimal). Nitrogenase activity was lost in about 3 h when cells were incubated aerobically in the dark. Re-exposure to light resulted in recovery of nitrogenase activity within 2 h. Blending, oxygen, or dark pre-incubation had similar effects upon cultures grown under air or nitrogen and did not inhibit light-dependent CO₂ fixation. We conclude that heterocysts are the sites of nitrogenase activity and propose a model for nitrogen fixation by Anabaena cylindrica.     

木麻黄和桤木离体根瘤和立地土壤的固氮酶与N2O还原酶活性的研究

为了解非豆科固氮树种的固氮酶和N_2O还原酶(Nos)活性,采用乙炔还原法和乙炔抑制技术对细枝木麻黄(Casuarina cunninghamiana)和江南桤木(Alnus trabeculosa)离体根瘤及立地土壤的两种酶活性进行了研究。结果表明,离体根瘤只在厌氧条件下有固氮酶活性,在好氧条件下有Nos活性。根瘤区根际土和非根瘤区根际土的固氮酶活性在好氧条件大于厌氧条件,Nos活性只表现在厌氧条件下。在好氧条件下,根瘤区根际土和非根瘤区根际土的固氮酶活性无显著差异;根瘤区根际土的Nos活性显著大于非根瘤区根际土。除离体根瘤在好氧条件下不表现固氮酶活性外,细枝木麻黄和桤木的离体根瘤、根瘤区根际土和非根瘤区根际土的固氮酶活性均都大于Nos活性。好氧条件下根瘤区根际土的固氮酶活性与非根瘤区根际土的呈极显著正相关,而厌氧条件下根瘤的固氮酶活性与好氧条件下根瘤区根际土和非根瘤区根际土固氮酶活性、好氧条件下根瘤的Nos活性与厌氧条件下根瘤区根际土和非根瘤区根际土Nos活性均呈极显著负相关。这为研究弗兰克氏菌结瘤植物共生固氮体系对N2O汇强度的影响和调控奠定基础。