Photosynthetic oxygen evolution within Sesbania rostrata stem nodules

The tropical wetland legume, Sesbania rostrata Brem. forms N₂-fixing nodules along its stem and on its roots after infection by Azorhizobium caulinodans . The N₂-fixing tissue is surrounded by a cortex of uninfected cells which, in the stem nodules (but not the root nodules), contain chloroplasts. The photosynthetic competence of these chloroplasts was assessed through a novel technique involving image analysis of chlorophyll a fluorescence. Calculation of the quantum efficiency of photosystem II (PS II) photochemistry from these images indicated that most of the chloroplasts with potential for non-cyclic photosynthetic electron transport were concentrated within the mid- and inner-cortex, close to the edge of the N₂-fixing tissue. PS II activity in the cortical cells was confirmed in vivo using O₂-specific microelectrodes which showed that the concentration of O₂ (pO₂) in the outer cortex could rise from less than 1% up to 23.4% upon increased irradiance of the nodule, but that the pO₂ of the inner cortex and infected tissue remained less than 0.0025%. Nitrogenase activity of stem nodules, as measured using a flow-through acetylene reduction assay (no H₂ evolution was evident), showed a reversible increase of 28% upon exposure of the nodules to supplemental light. This increase resembled that obtained with stem nodules upon their exposure to an external pO₂ of 40%.     

Isolation and identification of a N2-fixing zoogloea-forming bacterium from kallar grass histoplane

Semi-solid medium was used to isolate an aerobic, N₂-fixing (C₂H₂-reducing), H₂-utilizing bacterium from the roots of kallar grass ( Leptochloa fusca ). The organism was identified by morphological, cultural and biochemical characteristics. The N₂-fixing, zoogloeal floc-forming isolate described here is a new species.     

Divergence in symbiotic interactions between same genotypic PCR–RFLP Frankia strains and different Casuarinaceae species under natural conditions

The symbiotic interactions between Frankia strains and their associated plants from the Casuarinaceae under controlled conditions are well documented but little is known about these interactions under natural conditions. We explored the symbiotic interactions between eight genotypically characterized Frankia strains and five Casuarinaceae species in long-term field trials. Characterization of strains was performed using the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) for the nifD – nifK intergenic transcribed spacer (ITS) and 16S–23S ITS. Assessments of the symbiotic interactions were based on nodulation patterns using nodule dry weight and viability, and on actual N₂ fixation using the δ¹⁵N method. The PCR–RFLP patterns showed that the analyzed strains belonged to the same genotypic group (CeD group), regardless of the host species and environment of origin. The nodule viability index is introduced as a new tool to measure the viability of perennial nodules and to predict their effectiveness. The host Casuarinaceae species was a key factor influencing both the actual N₂-fixing activity of the associated Frankia strain and the viability of nodules within a location. This is the first study providing information on the symbiotic interactions between genotypically characterized Frankia strains and actinorhizal plants under natural conditions. The results revealed a way to improve a long-term management of the Casuarinaceae symbiosis.     

Effects of mycorrhizal colonization on biomass production and nitrogen fixation of black locust (Robinia pseudoacacia) seedlings grown under elevated atmospheric carbon dioxide

Interactive effects of elevated atmospheric CO₂ and arbuscular mycorrhizal (AM) fungi on biomass production and N₂ fixation were investigated using black locust ( Robinia pseudoacacia ). Seedlings were grown in growth chambers maintained at either 350 μmol mol⁻¹ or 710 μmol mol⁻¹ CO₂. Seedlings were inoculated with Rhizobium spp. and were grown with or without AM fungi. The ¹⁵N isotope dilution method was used to determine N source partitioning between N₂ fixation and inorganic fertilizer uptake. Elevated atmospheric CO₂ significantly increased the percentage of fine roots that were colonized by AM fungi. Mycorrhizal seedlings grown under elevated CO₂ had the greatest overall plant biomass production, nodulation, N and P content, and root N absorption. Additionally, elevated CO₂ levels enhanced nodule and root mass production, as well as N₂ fixation rates, of non- mycorrhizal seedlings. However, the relative response of biomass production to CO₂ enrichment was greater in non-mycorrhizal seedlings than in mycorrhizal seedlings. This study provides strong evidence that arbuscular mycorrhizal fungi play an important role in the extent to which plant nutrition of symbiotic N₂-fixing tree species is affected by enriched atmospheric CO₂.     

Effects of exogenous application and stem infusion of ascorbate on soybean (Glycine max) root nodules

Numerous biochemical and physiological studies have demonstrated the importance of ascorbate (ASC) as a reducing agent and antioxidant in higher plant metabolism. Of special note is the capacity of ASC to eliminate damaging activated oxygen species (AOS) including O₂^−· and H₂O₂. N₂-fixing legume nodules are especially vulnerable to oxidative damage because they contain large amounts of leghaemoglobin which produces AOS through spontaneous autoxidation; thus, ASC and other components of the ascorbate–reduced glutathione (ASC–GSH) pathway are critical antioxidants in nodules. In order to establish a meaningful correlation between concentrations of ASC and capacity for N₂ fixation in legume root nodules, soybean ( Glycine max ) plants were treated with excess ASC via exogenous irrigation or continuous intravascular infusion through needles inserted directly into plant stems. Treatment with ASC led to striking increases in nitrogenase activity (acetylene reduction), nodule leghaemoglobin content, and activity of ASC peroxidase, a key antioxidant enzyme. The concentration of lipid peroxides, which are indicators of oxidative damage and onset of senescence, was decreased in ASC-treated nodules. These results support the conclusion that ASC is critical for N₂ fixation and that elevated ASC allows nodules to maintain a greater capacity to fix N₂ over longer periods.     

Differences in nitrogen metabolism of Faidherbia albida and other N2-fixing tropical woody acacias reflect habitat water availability

The activities of nitrate reductase and glutamine synthetase were evaluated in young plants of Faidherbia albida , a tropical woody legume, fed with different N sources under hydroponic conditions. Results showed that assimilation of both NO₃⁻ and NH₄⁺ preferentially took place in shoots. A basal amount of nitrate reductase activity was detected in shoots of plants grown with an NO₃⁻-free solution or placed under N₂-fixing conditions, and also in nodules of N₂-fixing plants. This strongly suggests that constitutive nitrate reductase activity is present in these organs. Analyses of the soluble nitrogenous content showed that the major form of N in the different organs was α-amino acids (particularly amides), irrespective of the N status of the culture conditions. The same result was obtained for nodulated plants grown in local sandy soil. In this case, amide-N generally accounted for more than 40% of the total soluble N. This was especially true in nodules. Ureide-N never exceeded 9% of the total soluble N and did not appear to increase with increasing nodule nitrogenase activity. Amides were also predominant in three N₂-fixing Sahelian acacias ( Acacia seyal , A. nilotica and A. tortilis ), showing that F. albida does not differ from Sahelian Acacia in terms of the metabolism of fixed N. However, like another Sahelian acacia growing preferentially near water ( A. nilotica ), F. albida can be distinguished from acacias growing strictly in arid zones ( A. seyal and A. tortilis ) in terms of initial growth, water and nitrate management.     

Nitrogen fixation (N-15 dilution) with soybeans under Thai field conditions. IV. Effect of N addition and Bradyrhizobium japonicum inoculation in soils with indigenous B. japonicum populations

The effects of Bradyrhizobium japonicum inoculation and pre-plant additions of N fertilizer on soybean ( Glycine max L. Merrill) yields and levels of N₂ fixation were studied under field conditions at two sites in Thailand. Bacterial inoculants were composed of B. japonicum strains selected for high N₂ fixation levels in Thai soils. Nitrogen fertilizer addition rates used were from 0 to 250 kg N/ha in 50 kg N/ha increments. At the Chiang Mai site in northern Thailand, bacterial inoculation increased nodule weights on plants receiving 100 kg N/ha or less. Increases in nodule parameters due to inoculation were evident at 45 d after planting (DAP) but disappeared by 60 DAP. Addition of N fertilizers decreased the incidence of nodulation and sap ureide contents and decreased the contribution of N₂ fixation to the N content of plants at maturity as measured by N-15 isotope dilution methods. At the Kampang Saen site in central Thailand, bacterial inoculation had significant positive effects on nodule numbers and weights, ARA, sap ureide contents and levels of N₂ fixed as measured by N-15 isotope dilution methods. Addition of N fertilizers at this site also reduced the effectiveness of N₂-fixing symbioses. It was concluded that small additions of N fertilizer added before planting did not significantly decrease N₂ fixation levels, but did have a significant positive effect on plant growth. Larger N additions would reduce N₂ fixation levels in excess of the benefits of adding more N in chemical form.     

Oxygen and the regulation of nitrogen fixation in legume nodules

In N₂-fixing legume nodules, O₂ is required in large amounts for aerobic respiration, yet nitrogenase, the bacterial enzyme that fixes N₂, is O₂ labile. A high rate of O₂ consumptition and a cortical barrier to gas diffusion work together to maintain a low, non-inhibitory O₂ concentration in the central, infected zone of the nodule. At this low O₂ concentration, cytosolic leghemoglobin is required to facilitate the diffusion of O₂ through the infected cell to the bacteria. The resistance of the cortical diffusion barrier is variable and is used by legume nodules to regulate the O₂ concentration in the infected cells such that it limits aerobic respiration and N₂ fixation at all times. The resistance of the diffusion barrier and therefore the degree of O₂ limitation seems to be regulated in response to changes in the O₂ concentration of the central infected zone, the supply of phloem sap to the nodule, and the rate of N assimilation into the end products of fixation.     

Alteration of N nutrition in Myrica gale induces changes in nodule growth, nodule activity and amino acid composition

Changes in nodule growth and activity and in the concentrations of soluble N compounds in nodules, leaves and xylem sap under conditions of altered N nutrition in the actinorhizal plant Myrica gale L. are reported. Altering the N nutrition of symbiotic plants may alter the internal regulation of combined N which in turn may regulate nodule growth and activity. Flushing nodules daily with 100% O₂ caused a decline in amide concentration and an increase in nodule growth although plants had recovered some nitrogenase activity within 4 h of exposure to O₂. Samples of nodules, leaves and xylem sap were derivatized and amino acids identified and quantified using either reverse phase high performance liquid chromatography or gas chromatography-mass spectrometry in single ion monitoring mode. The ratio of asparagine in the nodules to that in the xylem was much higher in plants fed N (6.7 for NH⁺₄-fed and 8.3 for NO⁻₃-fed plants) than for N₂-fixing plants (2.5). Significant amounts of ¹⁵N added as ¹⁵NH⁺₄ or ¹⁵NO⁻₃ accumulated in nodules following accumulation in the shoot which is consistent with the translocation of N to the nodules via the phloem. The uptake of ¹⁵NH⁺₄ led to the synthesis and subsequent translocation of glutamine in the xylem sap. These results are discussed in terms of the feedback mechanisms that may regulate nitrogen fixation in Myrica root nodules.     

Plant regulated aspects of nodulation and N2 fixation

Abstract. Root nodule organogenesis is described. Plant regulated aspects of nodulation and N₂ fixation are reviewed and discussed. Since the effective N₂ fixing symbiosis requires the interaction of the host plant and bacterium in an appropriate environment (the rhizosphere and the root nodule) it is essential that research aimed at improving N₂ fixation involve a knowledge and understanding of the plant genes that affect nodule development, growth, and function. Current knowledge of host plant genes involved in N₂ fixation is summarized. Various experimental approaches to the study of the host plant's contribution to nodulation are noted. The functions of nodule specific proteins (nodulins) in symbiosis are delineated. Future areas of research are suggested.     

Carbon Costs Associated with N2 Fixation in Vicia faba L and Pisum sativum 1. over a 14-Day Period

Abstract: Long-term (14 days) carbon costs of N₂ fixation were studied in pot trials. For this purpose the CO₂ release from the root space of nodulated and non-nodulated (urea nourished) Vicia faba L. and Pisum sativum L. plants was compared and related to the amount of fixed or assimilated N. Additional measurements of shoot CO₂ exchange and dry matter increment were carried out in order to calculate the overall carbon balance. The carbon costs for N₂ fixation in Vicia faba 1. (2.87 mg C/mg N_fiX) were higher than in Pisum sativum L. (2.03 mg C/mg N_fix). However, the better carbon efficiency in Pisum sativum 1. did not lead to a better growth performance compared to Vicia faba L. Vicia faba L. compensated for the carbon and energy expenditure by more intensive photosynthesis in the N₂-fixing treatment. This was not the case with Pisum sativum L., where the carbon balance indicates that the carbon costs of N₂ fixation restricted root growth. It is proposed that low carbon costs for N₂ fixation indicate an adaptation to a critical carbon supply of roots and nodules, e.g., during the pod-filling of grain legumes.     

The role of phosphorus in nitrogen fixation by young pea plants (Pisum sativum)

The influence of P on N₂ fixation and dry matter production of young pea ( Pisum sativum L. cv. Bodil) plants grown in a soil-sand mixture was investigated in growth cabinet experiments. Nodule dry weight, specific C₂H₂ reduction and P concentration in shoots responded to P addition before any growth response could be observed. The P concentration in nodules responded only slightly to P addition. A supply of P to P-deficient plants increased both the nodule dry weight, specific C₂H₂ reduction and P concentration in shoots relatively faster than it increased shoot dry weight and P concentration in nodules. Combined N applied to plants when N₂ fixation had commenced, increased shoot dry weight only at the highest P levels. This indicates that the smaller plant growth at the low P levels did not result from N deficiency. The reduced nodulation and N₂ fixation in P-deficient plants seem to be caused by impaired shoot metabolism and not by a direct effect of P deficiency of the nodules.     

Nodule phosphoenolpyruvate carboxylase: a review

Recent data concerning the fixation of CO₂ and the functioning of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) in legume, nodules are reviewed. The activites of N₂ fixation (acctylene reduction) and PEP carboxylase are correlated Activities of PEP carboxylase are always higher in nodules than in roots. PEP carboxylase is located in the cytosol of the plant part of the nodules. When nodules are fed with ¹⁴CO₂, radioactivity appears predominantly in malate and aspartate. The resolution of isoenzymes of PEP carboxylase shows one more band in nodules than in related roots. The role of PEP carboxylase in nodule metabolism is discussed.     

Distribution of sym-homosperimidine in eubacteria, cyanobacteria, algae and ferns

Abstract Polyamines were analyzed in 12 of N₂-fixing aerobic eubacteria and other eubacteria, cyanobacteria, algae and ferns. sym -Homospermidine (homospermidine) was found to be widely distributed as a major polyamine in various N₂-fixing eubacteria which belong to Azospirillum, Agromonas, Beijerinckia, Bradyrhizobium, Rhizobium and Xathnbacter . 3 species of Azotobater contained spermidine but not homospermidine, though they are N₂-fixing eubactera. Homospermidine is also distributed in some eubacteria, i.e., the photosynthetic Rhodopseudomanas rutila and the sulfur-oxidizing Thiobacillus denitrificans , a cyanobacterium, Synechococcus sp., and in the cyanobacterium-symbiotic ferns, Azolla imbircatta and Azolla japonica .     

广西含羞草科和云实科一些植物结瘤状况的调查

调查了广西一些地区含羞草科植物34种,云实科植物39种,发现有瘤的植物分别为27种和6种,其中格木是首次报道有瘤,电子显微镜切片表明这些瘤是根瘤菌侵入所形成。     

The effect of potassium deficiency on growth and N2-fixation in Trifolium repens

The effects of potassium (K) deficiency on growth, N₂-fixation and photosynthesis in white clover ( Trifolium repens L.) were investigated using natural occurring gas fluxes on the nodules in real time of plants under three contrasting relative addition rates of K causing mild K deficiency, or following abrupt withdrawal of the K supply causing strong K deficiency of less than 0.65% in dry matter. A steady-state below-optimum K supply rate led to an increase in CO₂-fixation per unit leaf surface area as well as per plant leaf surface. However, nitrogenase activity per unit root weight and per unit nodule weight was maintained, as was the efficiency with which electrons were allocated to the reduction of N₂ in the nodules. Abrupt K removals stimulated nodule growth strongly without delay, but as K concentrations decreased in the plant tissue a significant decline in nitrogenase activity per unit root weight as well as per unit nodule mass occurred. Further, the rate of photosynthesis per unit leaf area was unaffected, while the CO₂ acquisition for the plant as a whole increased due to an expansion of total leaf area whereas the leaf area per unit leaf weight was unaffected. The ratio between CO₂-fixation and N₂-fixation increased, although not statistically significant, under short-term K deprivation as well as under long-term low K supply indicating a downregulation of nodule activity following morphological and growth adjustments. This downregulation took place despite a partly substitution of the K by Na. It is concluded that N₂-fixation does not limit the growth of K-deprived clover plants. K deprivation induces changes in the relative growth of roots, nodules, and shoots rather than changes in N and/or carbon uptake rates per unit mass or area of these organs.     

New type of N2-fixing unicellular cyanobacterium (blue-green alga)

Abstract A new N₂-fixing unicellular cyanobacterium identified as a Synechococcus sp. was isolated and purified as an axenic culture. It fixed N₂ aerobically either under continuous illumination or in alternating light-dark cycle. The N₂-fixing properties of the new isolate and Gloeocapsa are discussed.     

15N natural abundance during early and late succession in a middle-European dry acidic grassland

δ¹⁵N and total nitrogen content of above- and belowground tissues of 13 plant species from two successional stages (open pioneer community and ruderal grass stage) of a dry acidic grassland in Southern Germany were analysed, in order to evaluate whether resource use partitioning by niche separation and N input by N₂-fixing legumes are potential determinants for species coexistence and successional changes. Within each stage, plants from plots with different legume cover were compared. Soil inorganic N content, total plant biomass and δ¹⁵N values of bulk plant material were significantly lower in the pioneer stage than in the ruderal grass community. The observed δ¹⁵N differences were rather species- than site-specific. Within both stages, there were also species-specific differences in isotopic composition between above- and belowground plant dry matter. Species-specific δ¹⁵N signatures may theoretically be explained by (i) isotopic fractionation during microbial-mediated soil N transformations; (ii) isotopic fractionation during plant N uptake or fractionation during plant–mycorrhiza transfer processes; (iii) differences in metabolic pathways and isotopic fractionation within the plant; or (iv) partitioning of available N resources (or pools) among plant groups or differential use of the same resources by different species, which seems to be the most probable route in the present case. A significant influence of N₂-fixing legumes on the N balance of the surrounding plant community was not detectable. This was confirmed by the results of an independent in situ removal experiment, showing that after 3 years there were no measurable differences in the frequency distribution between plots with and without N₂-fixing legumes.     

Expression of nir, nor and nos denitrification genes from Bradyrhizobium japonicum in soybean root nodules

Expression of Bradyrhizobium japonicum wild-type strain USDA110 nirK , norC and nosZ denitrification genes in soybean root nodules was studied by in situ histochemical detection of β -galactosidase activity. Similarly, P_nirK- lacZ , P_norC- lacZ , and P_nosZ- lacZ fusions were also expressed in bacteroids isolated from root nodules. Levels of β -galactosidase activity were similar in both bacteroids and nodule sections from plants that were solely N₂-dependent or grown in the presence of 4 m M KNO₃. These findings suggest that oxygen, and not nitrate, is the main factor controlling expression of denitrification genes in soybean nodules. In plants not amended with nitrate, B. japonicum mutant strains GRK308, GRC131, and GRZ25, that were altered in the structural nirK , norC and nosZ genes, respectively, showed a wild-type phenotype with regard to nodule number and nodule dry weight as well as plant dry weight and nitrogen content. In the presence of 4 m M KNO₃, plants inoculated with either GRK308 or GRC131 showed less nodules, and lower plant dry weight and nitrogen content, relative to those of strains USDA110 and GRZ25. Taken together, the present results revealed that although not essential for nitrogen fixation, mutation of either the structural nirK or norC genes encoding respiratory nitrite reductase and nitric oxide reductase, respectively, confers B. japonicum reduced ability for nodulation in soybean plants grown with nitrate. Furthermore, because nodules formed by each the parental and mutant strains exhibited nitrogenase activity, it is possible that denitrification enzymes play a role in nodule formation rather than in nodule function.