Sinorhizobium meliloti succinylated high‐molecular‐weight succinoglycan and the Medicago truncatula LysM receptor‐like kinase MtLYK10 participate independently in symbiotic infection

The formation of nitrogen‐fixing nodules on legume hosts is a finely tuned process involving many components of both symbiotic partners. Production of the exopolysaccharide succinoglycan by the nitrogen‐fixing bacterium Sinorhizobium meliloti 1021 is needed for an effective symbiosis with Medicago spp., and the succinyl modification to this polysaccharide is critical. However, it is not known when succinoglycan intervenes in the symbiotic process, and it is not known whether the plant lysin‐motif receptor‐like kinase MtLYK10 intervenes in recognition of succinoglycan, as might be inferred from work on the Lotus japonicus MtLYK10 ortholog, LjEPR3. We studied the symbiotic infection phenotypes of S. meliloti mutants deficient in succinoglycan production or producing modified succinoglycan, in wild‐type Medicago truncatula plants and in Mtlyk10 mutant plants. On wild‐type plants, S. meliloti strains producing no succinoglycan or only unsuccinylated succinoglycan still induced nodule primordia and epidermal infections, but further progression of the symbiotic process was blocked. These S. meliloti mutants induced a more severe infection phenotype on Mtlyk10 mutant plants. Nodulation by succinoglycan‐defective strains was achieved by in trans rescue with a Nod factor‐deficient S. meliloti mutant. While the Nod factor‐deficient strain was always more abundant inside nodules, the succinoglycan‐deficient strain was more efficient than the strain producing only unsuccinylated succinoglycan. Together, these data show that succinylated succinoglycan is essential for infection thread formation in M. truncatula, and that MtLYK10 plays an important, but different role in this symbiotic process. These data also suggest that succinoglycan is more important than Nod factors for bacterial survival inside nodules.     

Rhizobial strain involvement in symbiosis efficiency of chickpea–rhizobia under drought stress: plant growth,nitrogen fixation and antioxidant enzyme activities

Chickpea plants were inoculated with two strains of Mesorhizobium ciceri: local strain (C-15) and non-local strain (CP-36) in order to evaluate plant growth parameters, activities of nitrogenase and antioxidant enzymes under drought stress as well as control condition within 15 days of imposition of drought stress. Biomass production, nodulation, nitrogen fixation and antioxidant enzyme activities under drought condition were compared. Under control condition, symbiotic efficiency in symbiosis formed by C-15 was higher than that in symbiosis derived by CP-36. Although drought stress decreased shoot dry weight, root dry weight, nodule dry weight and nitrogen fixation in both symbioses, the rate of decline in plants inoculated with CP-36 was higher than that in symbiosis chickpea with C-15. Therefore, symbioses showed different tolerance level under drought condition which was essentially correlated with symbiotic performance at non-stressful conditions. Under drought stress, nodular peroxidase (POX) activity increased in both symbioses but was higher in nodules produced by C-15. Ascorbate peroxidase (APX) increased significantly in nodules of symbiosis of chickpea with C-15. Catalase (CAT) and glutation reductase (GR) declined in both symbioses which decline extent in symbiosis with C-15 was lower than that in the nodules of CP-36. These results suggested contribution of rhizobial partner in enhancing the tolerance of symbioses to drought stress, which was related with the increase of antioxidant enzyme activities (APX and POX) under drought conditions.     

Lotus japonicus SUNERGOS1 encodes a predicted subunit A of a DNA topoisomerase VI that is required for nodule differentiation and accommodation of rhizobial infection

A symbiotic mutant of Lotus japonicus, called sunergos1‐1 (suner1‐1), originated from a har1‐1 suppressor screen. suner1‐1 supports epidermal infection by Mesorhizobium loti and initiates cell divisions for organogenesis of nodule primordia. However, these processes appear to be temporarily stalled early during symbiotic interaction, leading to a low nodule number phenotype. This defect is ephemeral and near wild‐type nodule numbers are reached by suner1‐1 at a later point after infection. Using an approach that combined map‐based cloning and next‐generation sequencing we have identified the causative mutation and show that the suner1‐1 phenotype is determined by a weak recessive allele, with the corresponding wild‐type SUNER1 locus encoding a predicted subunit A of a DNA topoisomerase VI. Our data suggest that at least one function of SUNER1 during symbiosis is to participate in endoreduplication, which is an essential step during normal differentiation of functional, nitrogen‐fixing nodules.     

Exploring cycad foliage as an archive of the isotopic composition of atmospheric nitrogen

Molecular nitrogen (N₂) constitutes the majority of Earth's modern atmosphere, contributing ~0.79 bar of partial pressure (pN₂). However, fluctuations in pN₂ may have occurred on 10⁷–10⁹ year timescales in Earth's past, perhaps altering the isotopic composition of atmospheric nitrogen. Here, we explore an archive that may record the isotopic composition of atmospheric N₂ in deep time: the foliage of cycads. Cycads are ancient gymnosperms that host symbiotic N₂‐fixing cyanobacteria in modified root structures known as coralloid roots. All extant species of cycads are known to host symbionts, suggesting that this N₂‐fixing capacity is perhaps ancestral, reaching back to the early history of cycads in the late Paleozoic. Therefore, if the process of microbial N₂ fixation records the δ¹⁵N value of atmospheric N₂ in cycad foliage, the fossil record of cycads may provide an archive of atmospheric δ¹⁵N values. To explore this potential proxy, we conducted a survey of wild cycads growing in a range of modern environments to determine whether cycad foliage reliably records the isotopic composition of atmospheric N₂. We find that neither biological nor environmental factors significantly influence the δ¹⁵N values of cycad foliage, suggesting that they provide a reasonably robust record of the δ¹⁵N of atmospheric N₂. Application of this proxy to the record of carbonaceous cycad fossils may not only help to constrain changes in atmospheric nitrogen isotope ratios since the late Paleozoic, but also could shed light on the antiquity of the N₂‐fixing symbiosis between cycads and cyanobacteria.     

Phytoglobins in the nuclei,cytoplasm and chloroplasts modulate nitric oxide signaling and interact with abscisic acid

Symbiotic hemoglobins provide O₂ to N₂‐fixing bacteria within legume nodules, but the functions of non‐symbiotic hemoglobins or phytoglobins (Glbs) are much less defined. Immunolabeling combined with confocal microscopy of the Glbs tagged at the C‐terminus with green fluorescent protein was used to determine their subcellular localizations in Arabidopsis and Lotus japonicus. Recombinant proteins were used to examine nitric oxide (NO) scavenging in vitro and transgenic plants to show S‐nitrosylation and other in vivo interactions with NO and abscisic acid (ABA) responses. We found that Glbs occur in the nuclei, chloroplasts and amyloplasts of both model plants, and also in the cytoplasm of Arabidopsis cells. The proteins show similar NO dioxygenase activities in vitro, are nitrosylated in Cys residues in vivo, and scavenge NO in the stomatal cells. The Cys/Ser mutation does not affect NO dioxygenase activity, and S‐nitrosylation does not significantly consume NO. We demonstrate an interaction between Glbs and ABA on several grounds: Glb1 and Glb2 scavenge NO produced in stomatal guard cells following ABA supply; plants overexpressing Glb1 show higher constitutive expression of the ABA responsive genes Responsive to ABA (RAB18), Responsive to Dehydration (RD29A) and Highly ABA‐Induced 2 (HAI2), and are more tolerant to dehydration; and ABA strongly upregulates class 1 Glbs. We conclude that Glbs modulate NO and interact with ABA in crucial physiological processes such as the plant's response to dessication.     

Long‐term non‐invasive and continuous measurements of legume nodule activity

Symbiotic nitrogen fixation is a process of considerable economic, ecological and scientific interest. The central enzyme nitrogenase reduces H⁺ alongside N₂, and the evolving H₂ allows a continuous and non‐invasive in vivo measurement of nitrogenase activity. The objective of this study was to show that an elaborated set‐up providing such measurements for periods as long as several weeks will produce specific insight into the nodule activity's dependence on environmental conditions and genotype features. A system was developed that allows the air‐proof separation of a root/nodule and a shoot compartment. H₂ evolution in the root/nodule compartment can be monitored continuously. Nutrient solution composition, temperature, CO₂ concentration and humidity around the shoots can concomitantly be maintained and manipulated. Medicago truncatula plants showed vigorous growth in the system when relying on nitrogen fixation. The set‐up was able to provide specific insights into nitrogen fixation. For example, nodule activity depended on the temperature in their surroundings, but not on temperature or light around shoots. Increased temperature around the nodules was able to induce higher nodule activity in darkness versus light around shoots for a period of as long as 8 h. Conditions that affected the N demand of the shoots (ammonium application, Mg or P depletion, super numeric nodules) induced consistent and complex daily rhythms in nodule activity. It was shown that long‐term continuous measurements of nodule activity could be useful for revealing special features in mutants and could be of importance when synchronizing nodule harvests for complex analysis of their metabolic status.     

Symbiotic Effectiveness and Host-Strain Interactions of Rhizobium fredii USDA 191 on Different Soybean Cultivars

Nodulation, acetylene reduction activity, dry matter accumulation, and total nitrogen accumulation by nodulated plants growing in a nitrogen-free culture system were used to compare the symbiotic effectiveness of the fast-growing Rhizobium fredii USDA 191 with that of the slow-growing Bradyrhizobium japonicum USDA 110 in symbiosis with five soybean (Glycine max (L.) Merr.) cultivars. Measurement of the amount of nitrogen accumulated during a 20-day period of vegetative growth (28 to 48 days after transplanting) showed that USDA 110 fixed 3.7, 39.1, 4.6, and 57.3 times more N₂ than did USDA 191 with cultivars Pickett 71, Harosoy 63, Lee, and Ransom as host plants, respectively. With the unimproved Peking cultivar as the host plant, USDA 191 fixed 3.3 times more N₂ than did the USDA 110 during the 20-day period. The superior N₂ fixation capability of USDA 110 with the four North American cultivars as hosts resulted primarily from higher nitrogenase activity per unit nodule mass (specific acetylene reduction activity) and higher nodule mass per plant. The higher N₂-fixation capability of USDA 191 with the Peking cultivar as host resulted primarily from higher nodule mass per plant, which was associated with higher nodule numbers. There was significant variation in the N₂-fixation capabilities of the four North American cultivar-USDA 191 symbioses. Pickett 71 and Lee cultivars fixed significantly more N₂ in symbiosis with USDA 191 than did the Harosoy 63 and Ransom cultivars. This quantitative variation in N₂-fixation capability suggests that the total incompatibility (effectiveness of nodulation and efficiency of N₂ fixation) of host soybean plants and R. fredii strains is regulated by more than one host plant gene. These results indicate that it would not be prudent to introduce R. fredii strains into North American agricultural systems until more efficient N₂-fixing symbioses between North American cultivars and these fast-growing strains can be developed. When inoculum containing equal numbers of USDA 191 and of strain USDA 110 was applied to the unimproved Peking cultivar in Perlite pot culture, 85% of the 160 nodules tested were occupied by USDA 191. With Lee and Ransom cultivars, 99 and 85% of 140 and 96 nodules tested, respectively, were occupied by USDA 110.     

Two Lotus japonicus symbiosis mutants impaired at distinct steps of arbuscule development

Arbuscular mycorrhiza (AM) fungi form nutrient‐acquiring symbioses with the majority of higher plants. Nutrient exchange occurs via arbuscules, highly branched hyphal structures that are formed within root cortical cells. With a view to identifying host genes involved in AM development, we isolated Lotus japonicus AM‐defective mutants via a microscopic screen of an ethyl methanesulfonate‐mutagenized population. A standardized mapping procedure was developed that facilitated positioning of the defective loci on the genetic map of L. japonicus, and, in five cases, allowed identification of mutants of known symbiotic genes. Two additional mutants representing independent loci did not form mature arbuscules during symbiosis with two divergent AM fungal species, but exhibited signs of premature arbuscule arrest or senescence. Marker gene expression patterns indicated that the two mutants are affected in distinct steps of arbuscule development. Both mutants formed wild‐type‐like root nodules upon inoculation with Mesorhizobium loti, indicating that the mutated loci are essential during AM but not during root nodule symbiosis.     

Peribacteroid space acidification: a marker of mature bacteroid functioning in Medicago truncatula nodules

Legumes form a symbiotic interaction with Rhizobiaceae bacteria, which differentiate into nitrogen‐fixing bacteroids within nodules. Here, we investigated in vivo the pH of the peribacteroid space (PBS) surrounding the bacteroid and pH variation throughout symbiosis. In vivo confocal microscopy investigations, using acidotropic probes, demonstrated the acidic state of the PBS. In planta analysis of nodule senescence induced by distinct biological processes drastically increased PBS pH in the N₂‐fixing zone (zone III). Therefore, the PBS acidification observed in mature bacteroids can be considered as a marker of bacteroid N₂ fixation. Using a pH‐sensitive ratiometric probe, PBS pH was measured in vivo during the whole symbiotic process. We showed a progressive acidification of the PBS from the bacteroid release up to the onset of N₂ fixation. Genetic and pharmacological approaches were conducted and led to disruption of the PBS acidification. Altogether, our findings shed light on the role of PBS pH of mature bacteroids in nodule functioning, providing new tools to monitor in vivo bacteroid physiology.     

Host adaptation and unexpected symbiont partners enable reef‐building corals to tolerate extreme temperatures

Understanding the potential for coral adaptation to warming seas is complicated by interactions between symbiotic partners that define stress responses and the difficulties of tracking selection in natural populations. To overcome these challenges, we characterized the contribution of both animal host and symbiotic algae to thermal tolerance in corals that have already experienced considerable warming on par with end‐of‐century projections for most coral reefs. Thermal responses in Platygyra daedalea corals from the hot Persian Gulf where summer temperatures reach 36°C were compared with conspecifics from the milder Sea of Oman. Persian Gulf corals had higher rates of survival at elevated temperatures (33 and 36°C) in both the nonsymbiotic larval stage (32–49% higher) and the symbiotic adult life stage (51% higher). Additionally, Persian Gulf hosts had fixed greater potential to mitigate oxidative stress (31–49% higher) and their Symbiodinium partners had better retention of photosynthetic performance under elevated temperature (up to 161% higher). Superior thermal tolerance of Persian Gulf vs. Sea of Oman corals was maintained after 6‐month acclimatization to a common ambient environment and was underpinned by genetic divergence in both the coral host and symbiotic algae. In P. daedalea host samples, genomewide SNP variation clustered into two discrete groups corresponding with Persian Gulf and Sea of Oman sites. Symbiodinium within host tissues predominantly belonged to ITS2 rDNA type C3 in the Persian Gulf and type D1a in the Sea of Oman contradicting patterns of Symbiodinium thermal tolerance from other regions. Our findings provide evidence that genetic adaptation of both host and Symbiodinium has enabled corals to cope with extreme temperatures in the Persian Gulf. Thus, the persistence of coral populations under continued warming will likely be determined by evolutionary rates in both, rather than single, symbiotic partners.     

Loss‐of‐function of ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (APN1) in Lotus japonicus restricts efficient nitrogen‐fixing symbiosis with specific Mesorhizobium loti strains

The nitrogen‐fixing symbiosis of legumes and Rhizobium bacteria is established by complex interactions between the two symbiotic partners. Legume Fix^– mutants form apparently normal nodules with endosymbiotic rhizobia but fail to induce rhizobial nitrogen fixation. These mutants are useful for identifying the legume genes involved in the interactions essential for symbiotic nitrogen fixation. We describe here a Fix^– mutant of Lotus japonicus, apn1, which showed a very specific symbiotic phenotype. It formed ineffective nodules when inoculated with the Mesorhizobium loti strain TONO. In these nodules, infected cells disintegrated and successively became necrotic, indicating premature senescence typical of Fix^– mutants. However, it formed effective nodules when inoculated with the M. loti strain MAFF303099. Among nine different M. loti strains tested, four formed ineffective nodules and five formed effective nodules on apn1 roots. The identified causal gene, ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (LjAPN1), encodes a nepenthesin‐type aspartic peptidase. The well characterized Arabidopsis aspartic peptidase CDR1 could complement the strain‐specific Fix^– phenotype of apn1. LjAPN1 is a typical late nodulin; its gene expression was exclusively induced during nodule development. LjAPN1 was most abundantly expressed in the infected cells in the nodules. Our findings indicate that LjAPN1 is required for the development and persistence of functional (nitrogen‐fixing) symbiosis in a rhizobial strain‐dependent manner, and thus determines compatibility between M. loti and L. japonicus at the level of nitrogen fixation.     

Immunogold localization of nodule-enhanced phosphoenolpyruvate carboxylase in alfalfa

Phosphoenolpyruvate carboxylase (PEPC; EC 4-1-1-31) plays a paramount role in providing carbon for synthesis of malate and aspartate in alfalfa (Medicago sativa L.) root nodules. PEPC protein and activity levels are highly enhanced in N₂-fixing alfalfa nodules. To ascertain the relationship between the cellular location of PEPC and root nodule metabolism, enzyme localization was evaluated by immunogold cytochemistry using alfalfa nodule PEPC antibodies. Gold labelling patterns in effective nodules showed that PEPC is a cytosolic enzyme and is distributed relatively equally in infected and uninfected cells of the nodule symbiotic zone. A high amount of labelling was also observed in pericycle cells of the nodule vascular system. Labelling was also detected within inner cortical cells, but the density was reduced by 60%. When Lotus corniculatus was transformed with a chimeric gene consisting of the 5′-upstream region of the PEPC gene fused to β-glucuronidase (GUS), GUS staining in nodules was consistent with immunogold localization patterns. The occurrence of PEPC in both infected and uninfected cells of the symbiotic zone of effective nodules coupled to the reduced amounts in ineffective nodules suggests a direct role for this enzyme in supporting N₂-fixation. PEPC localization in the uninfected, interstitial cells of the symbiotic zone indicates that these cells may also have a role in nodule carbon metabolism. Moreover, the association of PEPC with the nodule vascular system implies a role for the enzyme in the transport of assimilates to and from the shoot.     

Measuring carbon and N2 fixation in field populations of colonial and free‐living unicellular cyanobacteria using nanometer‐scale secondary ion mass spectrometry1

Unicellular cyanobacteria are now recognized as important to the marine N and C cycles in open ocean gyres, yet there are few direct in situ measurements of their activities. Using a high‐resolution nanometer scale secondary ion mass spectrometer (nanoSIMS), single cell N₂ and C fixation rates were estimated for unicellular cyanobacteria resembling N₂ fixer Crocosphaera watsonii. Crocosphaera watsonii‐like cells were observed in the subtropical North Pacific gyre (22°45′ N, 158°0′ W) as 2 different phenotypes: colonial and free‐living. Colonies containing 3–242 cells per colony were observed and cell density in colonies increased with incubation time. Estimated C fixation rates were similarly high in both phenotypes and unexpectedly for unicellular cyanobacteria 85% of the colonial cells incubated during midday were also enriched in ¹⁵N above natural abundance. Highest ¹⁵N enrichment and N₂ fixation rates were found in cells incubated overnight where up to 64% of the total daily fixed N in the upper surface waters was attributed to both phenotypes. The colonial cells retained newly fixed C in a sulfur‐rich matrix surrounding the cells and often cells of both phenotypes possessed areas (<1 nm) of enriched ¹⁵N and ¹³C resembling storage granules. The nanoSIMS imaging of the colonial cells also showed evidence for a division of N₂ and C fixation activity across the colony where few individual cells (<34%) in a given colony were enriched in both ¹⁵N and ¹³C above the colony average. Our results provide new insights into the ecophysiology of unicellular cyanobacteria.     

Lotus SHAGGY‐like kinase 1 is required to suppress nodulation in Lotus japonicus

Glycogen synthase kinase/SHAGGY‐like kinases (SKs) are a highly conserved family of signaling proteins that participate in many developmental, cell‐differentiation, and metabolic signaling pathways in plants and animals. Here, we investigate the involvement of SKs in legume nodulation, a process requiring the integration of multiple signaling pathways. We describe a group of SKs in the model legume Lotus japonicus (LSKs), two of which respond to inoculation with the symbiotic nitrogen‐fixing bacterium Mesorhizobium loti. RNAi knock‐down plants and an insertion mutant for one of these genes, LSK1, display increased nodulation. Ηairy‐root lines overexpressing LSK1 form only marginally fewer mature nodules compared with controls. The expression levels of genes involved in the autoregulation of nodulation (AON) mechanism are affected in LSK1 knock‐down plants at low nitrate levels, both at early and late stages of nodulation. At higher levels of nitrate, these same plants show the opposite expression pattern of AON‐related genes and lose the hypernodulation phenotype. Our findings reveal an additional role for the versatile SK gene family in integrating the signaling pathways governing legume nodulation, and pave the way for further study of their functions in legumes.     

Lotus japonicus NOOT‐BOP‐COCH‐LIKE1 is essential for nodule,nectary, leaf and flower development

The NOOT‐BOP‐COCH‐LIKE (NBCL) genes are orthologs of Arabidopsis thaliana BLADE‐ON‐PETIOLE1/2. The NBCLs are developmental regulators essential for plant shaping, mainly through the regulation of organ boundaries, the promotion of lateral organ differentiation and the acquisition of organ identity. In addition to their roles in leaf, stipule and flower development, NBCLs are required for maintaining the identity of indeterminate nitrogen‐fixing nodules with persistent meristems in legumes. In legumes forming determinate nodules, without persistent meristem, the roles of NBCL genes are not known. We thus investigated the role of Lotus japonicus NOOT‐BOP‐COCH‐LIKE1 (LjNBCL1) in determinate nodule identity and studied its functions in aerial organ development using LORE1 insertional mutants and RNA interference‐mediated silencing approaches. In Lotus, LjNBCL1 is involved in leaf patterning and participates in the regulation of axillary outgrowth. Wild‐type Lotus leaves are composed of five leaflets and possess a pair of nectaries at the leaf axil. Legumes such as pea and Medicago have a pair of stipules, rather than nectaries, at the base of their leaves. In Ljnbcl1, nectary development is abolished, demonstrating that nectaries and stipules share a common evolutionary origin. In addition, ectopic roots arising from nodule vascular meristems and reorganization of the nodule vascular bundle vessels were observed on Ljnbcl1 nodules. This demonstrates that NBCL functions are conserved in both indeterminate and determinate nodules through the maintenance of nodule vascular bundle identity. In contrast to its role in floral patterning described in other plants, LjNBCL1 appears essential for the development of both secondary inflorescence meristem and floral meristem.     

Matching population diversity of rhizobial nodA and legume NFR5 genes in plant–microbe symbiosis

We hypothesized that population diversities of partners in nitrogen‐fixing rhizobium–legume symbiosis can be matched for “interplaying” genes. We tested this hypothesis using data on nucleotide polymorphism of symbiotic genes encoding two components of the plant–bacteria signaling system: (a) the rhizobial nodA acyltransferase involved in the fatty acid tail decoration of the Nod factor (signaling molecule); (b) the plant NFR5 receptor required for Nod factor binding. We collected three wild‐growing legume species together with soil samples adjacent to the roots from one large 25‐year fallow: Vicia sativa, Lathyrus pratensis, and Trifolium hybridum nodulated by one of the two Rhizobium leguminosarum biovars (viciae and trifolii). For each plant species, we prepared three pools for DNA extraction and further sequencing: the plant pool (30 plant indiv.), the nodule pool (90 nodules), and the soil pool (30 samples). We observed the following statistically significant conclusions: (a) a monotonic relationship between the diversity in the plant NFR5 gene pools and the nodule rhizobial nodA gene pools; (b) higher topological similarity of the NFR5 gene tree with the nodA gene tree of the nodule pool, than with the nodA gene tree of the soil pool. Both nonsynonymous diversity and Tajima's D were increased in the nodule pools compared with the soil pools, consistent with relaxation of negative selection and/or admixture of balancing selection. We propose that the observed genetic concordance between NFR5 gene pools and nodule nodA gene pools arises from the selection of particular genotypes of the nodA gene by the host plant.     

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。