Dinitrogenase reductase (Fe-protein) of nitrogenase in the cyanobacterial symbionts of three Azolla species: Localization and sequence of appearance during heterocyst differentiation

Transmission electron microscopy and immunocytological labeling were used to study the distribution and ontological occurrence of dinitrogenase reductase (Fe-protein) of nitrogenase in cyanobacterial symbionts within young leaves of the water-ferns Azolla filiculoides Lamarck, A. caroliniana Willdenow, and A. pinnata R. Brown. Rabbit anti-dinitrogenase reductase antisera and goat anti-rabbit-immunoglobulin G antibody conjugated to colloidal gold were used as probes. Western blot analyses showed that a polypeptide of approx. 36 kDa (kdalton) was recognized in the symbionts of all three Azolla species and that the polyclonal sera used were monospecific. In all symbionts, nitrogenase was immunologically recognizable within heterocysts. It was absent from vegetative cells, and also from the akinetes of the A. caroliniana and A. pinnata symbionts. The differentiation of vegetative cells into heterocysts in all three symbionts was initiated by formation of additional external cell-wall layers and narrowing of the neck followed by loss of glycogen, mild vesiculation of thylakoid membranes, and the appearance of polar nodules. No nitrogenase was detected at these early stages, but it appeared in the intermediate proheterocyst stage concomitantly with the formation of contorted membranes, and reached the strongest labeling in mature heterocysts, containing extensive tightly packed membranes. Nitrogenase was evenly distributed throughout heterocysts except at the polar regions, which contained honey-comb configurations and large polar nodules. With increased age of the A. caroliniana and A. pinnata symbionts, heterocysts became highly vesiculated, with a concomitant decrease in the amount of nitrogenase detected.Abbreviations IgG Immunoglobulin G - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TEM transmission electron micrograph     

Nodulation and growth response ofAllocasuarina andCasuarina species to phosphorus fertilization

To examine how soil phosphorus status affects nitrogen fixation by the Casuarinaceae —Frankia symbiosis,Casuarina equisetifolia and two species ofAllocasuarina (A. torulosa andA. littoralis) inoculated or fertilized with KNO₃ were grown in pots in an acid soil at 4 soil phosphate levels. InoculatedC. equisetifolia nodulated well by 12 weeks after planting and the numbers and weight of nodules increased markedly with phosphorus addition. Growth ofC. equisetifolia dependent on symbiotically fixed nitrogen was more sensitive to low levels of phosphorus (30 mg kg^–1 soil) than was growth of seedings supplied with combined nitrogen; at higher levels of phosphorus, the growth response curves were similar for both nitrogen fertilized and inoculated plants. The interaction between phosphorus and nitrogen treatments (inoculated and nitrogen fertilized) demonstrated that there was a greater requirement of phosphorus for symbiotic nitrogen fixation than for plant growth when soil phosphorus was low.WithAllocasuarina species, large plant to plant variation in nodulation occurred both within pots and between replicates. This result suggests genetic variation in nodulation withinAllocasuarina species. Nodulation ofAllocasuarina species did not start until 16 weeks after planting and no growth response due toFrankia inoculation was obtained at the time of harvest. Addition of nitrogen starter is suggested to boost plant growth before the establishment of the symbiosis. Growth ofAllocasuarina species fertilized with nitrogen responded to increasing levels of phosphorus up to 90 mg P/kg soil after which it declined by 69% forA. littoralis. The decrease in shoot weight ofA. littoralis, A. torulosa, C. equisetifolia andC. cunninghamiana at high phosphorus was confirmed in a sand culture experiment, and may be atributable to phosphorus toxicity.     

Canavanine-lnduced inhibition of growth and heterocyst differentiation inAnabaena doliolum and isolation of a canavanine-resistant mutant

The effect of the arginine analogue, canavanine on growth and heterocyst differentiation in the nitrogen-fixing algaAnabaena doliolum has been studied. The analogue inhibited growth and heterocyst differentiation at a concentration as low as 1 μM. The treated algal cells lacked conspicuous granular inclusions, whereas treatment with chloramphenicol led to increased synthesis of granules (probably cyanophycin granules). Exogenously added arginine completely reversed the effect of the analogue but lysine could only partially relieve the effect. A time course study with canavanine indicated inhibition of fresh protein(s) synthesis at all steps where a new class of proteins is synthesized so that the action of the analogue does not seem to be specific for a particular kind of protein. A mutant resistant to this analogue has been successfully isolated indicating that this alga does not show mutational immunity at least to the amino acid analogues unlike in the observation with different antibiotics. Our observations indicate that canavanine either directly inhibits protein synthesis or forms defective protein(s) which produces all the observed effects.     

Influence of nitrogen and phosphorus fertilizers on ammonia-assimilating enzymes of Azolla

Summary A field experiment was conducted and studied the effect of nitrogen and phosphorus on ammonia assimilating enzymes of Azolla. Nitrogen and phosphorus at 30 and 60 kg/ha respectively were tested andAzolla pinnata was inoculated at 200 g/m². The Azolla samples were drawn on 24th hr, 7th day and 14th day and the ammonia assimilating enzymes glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamine dehydrogenase (GDH) were estimated. Nitrogen and phosphorus have markedly suppressed the GDH activity but fertilizer nitrogen has no significant influence in inhibiting the enzyme activity of GOGAT and GS. In general phosphorus application also has stimulated the GS activity significantly during the first sampling period of 24th hour.     

Interactive effects of exogenous combined nitrogen and phosphorus on growth and nitrogen fixation by azolla

Effects of N source and media-N and P levels were examined on growth, N uptake, and N₂ fixation ofAzolla pinnata withAnabaena azollae association (azolla) at two inoculum-P concentrations. Each expeiment was conducted for 7 days in a growth chamber using azolla at a predetermined inoculum-P concentration and the growth media containing a combination of four levels of P (0, 15, 75, and 200 M) and three levels (0, 1, and 5 mM) of either¹⁵N-enriched NH ₄ ⁺ as ammonium sulfate or¹⁵N-enriched NO ₃ ^– as potassium nitrate. Nitrogen uptake and N₂ fixation were measured by¹⁵N isotopic dilution method. Tissue P and N, N uptake, and N₂-fixation increased with increasing P concentration in the media regardless of the inoculum-P level of azolla. Increasing P concentration in the media increased growth of azolla at low inoculum P, but the effect on high inoculum-P azolla was either small or absen. High inoculum-P concentration resulted in increased growth, tissue-N and P concentrations, N uptake, and N₂ fixation by azolla. Ammonium in the growth media caused larger increase in tissue-N and greater repression of N₂ fixation than equimolar concentration of NO ₃ ^– . In the presence of NH ₄ ⁺ or NO ₃ ^– , in the growth media, N uptake by azolla exceeded the corresponding decrease in N₂ fixation, resulting in an overall increase in tissue-N concentration. Phosphorus in the media tended to negate the inhibitory effect of NH ₄ ⁺ or NO ₃ ^– on N₂ fixation. A multiple regression model showed that the effect of tissue-N on N₂ fixation was negative while that of tissue-P was positive. Therefore, a relative change in tissue-N and P appeared to regulate N₂ fixation. Tissue-N and P had similar effects on relative growth rate of azolla also. Inoculum-P level of azolla was important in determining the response to media-P.This research was supported by a grant from USAID under Indo-US Science and Technology Initiative.     

Oxygen concentration, nitrogenase activity and heterocyst frequency in the leaf cavities of Azolla filiculoides Lam

Following exposure to UV light the synthesis of six polypeptides (112, 100, 89, 76, 71 and 65 kDa) was found to be enhanced or induced in the alga Chlamydomonas reinhardtii. Treatment with 4-nitroquinoline-N-oxide (NQO) resulted in the enhanced/induced synthesis of six polypeptides with molecular masses similar to those enhanced following exposure to UV light. Heat shock resulted in the enhanced synthesis of five polypeptides (89, 76, 71, 60 and 22 kDa), three of which (89, 76 and 71 kDa) had apparently identical mobilities to polypeptides whose synthesis was enhanced following UV treatment.     

施盐和磷对台湾相思幼苗光合作用及养分特征的影响

为探究施盐和磷对重要海防林树种台湾相思幼苗叶光合作用与养分特征的影响,该研究设置0%(B₀)、0.2%(B₁)、0.4%(B₂)、0.6%(B₃)、0.8%(B₄)的NaCl溶液和0 (P₀)、0.5 (P₁)、1.5 (P₂)g·kg^-1 3个供磷水平的过磷酸钙磷肥,在此基础上设置盐磷6个耦合处理,测定幼苗光合作用和养分特征指标。结果表明：(1)盐胁迫显著抑制台湾相思幼苗的生长发育,盐含量越高影响程度越大;低盐施磷对台湾相思幼苗生长不利,中高盐施磷显著减缓盐对幼苗生长的抑制作用。(2)台湾相思幼苗光合作用受盐胁迫影响显著;中低盐施磷后气孔关闭程度上升会加剧盐胁迫对幼苗光合作用的影响,高盐适当施磷可显著提高台湾相思幼苗光合能力。(3)盐胁迫显著降低叶绿素含量且对光系统Ⅱ造成危害;低盐胁迫施磷对台湾相思幼苗叶绿素合成不利,高盐适当施磷可以提高叶绿素合成量、稳定细胞膜结构以及提高叶片潜在光合能力...     

Extracellular polypeptides ofAnabaena L-31: Evidence for their role in regulation of heterocyst formation

Extracellular polypeptides released by both N₂-grown [peptide I] and NO₃-grown [peptide II]Anabaena L-31 have molecular weight of approximately 3,500 but have distinctly different amino acid composition. Acid hydrolysis of the peptide I fraction (obtained by separation on Sephadex G-25) yielded ten amino acids whereas that from peptide II fraction yielded only 3 amino acids. On addition to a freshly inoculated N₂-grown culture, the peptide I fraction stimulated pro-heterocyst and to a lesser extent heterocyst differentiation, whereas the peptide II fraction strongly inhibited differentiation. The inhibitory effect of polypeptide II fraction could not be relieved by methionine sulphoximine, which by itself enhances differentiation, but was greatly relieved by addition of the peptide I fraction. The data suggest but does not prove, thatAnabaena L-31 synthesises “inducer” or “inhibitor” peptides which could possibly control pattern formation.     

Evidence for an ammonium transport system in free-living and symbiotic cyanobacteria

The free-living cyanobacterium Anabaena variabilis showed a biphasic pattern of ¹⁴CH₃NH ₃ ⁺ uptake. Initial accumulation (up to 60 s) was independent of CH₃NH ₃ ⁺ metabolism, but long-term uptake was dependent on its metabolism via glutamine synthetase (GS). The CH₃NH ₃ ⁺ was converted into methylglutamine which was not further metabolised. The addition of l-methionine-dl-sulphoximine (MSX), to inhibit GS, inhibited CH₃NH ₃ ⁺ metabolism, but did not affect the CH₃NH ₃ ⁺ transport system.NH ₄ ⁺ , when added after the addition of ¹⁴CH₃NH ₃ ⁺ , caused the efflux of free CH₃NH ₃ ⁺ ; when added before ¹⁴CH₃NH ₃ ⁺ , NH ₄ ⁺ inhibited its uptake indicating that both NH ₄ ⁺ and CH₃NH ₃ ⁺ share a common transport system. Carbonylcyanide m-chlorophenylhydrazone and triphenyl-methylphosphonium both inhibited CH₃NH ₃ ⁺ accumulation indicating that the transport system was -dependent. At pH 7 and at an external CH₃NH ₃ ⁺ concentration of 30 mol dm^-3, A. variabilis showed a 40-fold intracellular accumulation of CH₃NH ₃ ⁺ (internal concentration 1.4 mmol dm^-3). Packets of the symbiotic cyanobacterium Anabaena azollae, directly isolated from the water fern Azolla caroliniana, also showed a -dependent NH ₄ ⁺ transport system suggesting that the reduced inhibitory effect of NH ₄ ⁺ on nitrogenase cannot be attributed to the absence of an NH ₄ ⁺ transport system but is probably related to the reduced GS activity of the cyanobiont.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - GS glutamine synthetase - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid - MSX l-methionine-dl-sulphoximine - membrane potential - pH transmembrane pH difference - TPMP⁺ triphenylmethylphosphonium     

Effects of plant species on phosphorus availability in a range of grassland soils

Vegetative conversion from grass to forest may influence soil nutrient dynamics and availability. A short-term (40 weeks) glasshouse experiment was carried out to investigate the impacts of ryegrass (Lolium perenne) and radiata pine (Pinus radiata) on soil phosphorus (P) availability in 15 grassland soils collected across New Zealand using ³³P isotopic exchange kinetics (IEK) and chemical extraction methods. Results from this study showed that radiata pine took up more P (4.5–33.5 mg P pot^–1) than ryegrass (1.1–15.6 mg pot^–1) from the soil except in the Temuka soil in which the level of available P (e.g., E _1min P_i, bicarbonate extractable P_i) was very high. Radiata pine tended to be better able to access different forms of soil P, compared with ryegrass. There were no significant differences in the level of water soluble P (Cp, intensity factor) between soils under ryegrass and radiata pine, but the levels of Cp were generally lower compared with original soils due to plant uptake. The growth of both ryegrass and radiata pine resulted in the redistribution of soil P from the slowly exchangeable P_i pool (E _{> 10m} P_i, reduced by 31.8% on the average) to the rapidly exchangeable P_i (E _1min-1d P_i, E _1d-10m P_i) pools in most soils. The values of R/r ₁ (the capacity factor) were also generally greater in most soils under radiata pine compared with ryegrass. Specific P mineralisation rates were significantly greater for soils under radiata pine (8.4–21.9%) compared with ryegrass (0.5–10.8%), indicating that the growth of radiata pine enhanced mineralisation of soil organic P. This may partly be ascribed to greater root phosphatase activity for radiata pine than for ryegrass. Plant species × soil type interactions for most soil variables measured indicate that the impacts of plant species on soil P dynamics was strongly influenced by soil properties.     

Endophyte transmission and activity in the Anabaena-Azolla association

Summary The survival of Azolla was studied in an artificial system which simulated the soil/water interface and the desiccation of soil during a fallow period in lowland rice culture. Tests with non-sporulating and sporulating Azolla fronds showed that Azolla only survives with sporulated fronds. At their reappearance the Azolla fronds already harboured the Anabaena endophyte. A detailed light microscopic and transmission electron microscopic study of macro- and micros-porocarp formation and development revealed that the endophyte is transmitted by the macrosporocarps and not by the microsporocarps. The Anabaena cells within the macrosporocarps are found just below the indusium cap. These cells are not nitrogen-fixing akinetes. The free-living Anabaena cells at the stem apex and below the overarching developing leaves do not bear heterocysts and accordingly are non nitrogen-fixing. During the development of the leaf the Anabaena enters the leaf cavity, but later the pore of this, cavity closes and the imprisoned cyanobacteria are lysed before the leaf decays. As the Azolla leaves age a nitrogen-fixing capability is successively built up concomittantly with the production of heterocysts. Heterocyst frequencies of 40–50% can be found inAnabaena azollae. Usually a gradient of nitrogen-fixing capacity occurs along the Azolla rhizome with two distinct peaks at leaf number 7/8 and at leaf number 13/14 from the apex.     

施磷对川西北高寒草地土壤磷形态及有效性的影响

以川西北高寒草地为研究对象,采用随机区组设计,设置0、10、20、30、40、50、60 g/m²的过磷酸钙(P₂O₅,16%)施肥试验,分析土壤不同形态磷含量和有效磷(Olsen-P)含量变化特征,探究施磷对川西北高寒草地土壤磷形态及有效磷的影响。结果表明：(1)随施磷量增加,土壤总磷(TP)含量先增加后趋于平稳而Olsen-P含量减少。高水平(50、60 g/m²)施磷下氢氧化钠有机磷(NaOH-Po)及残留磷(Residual-P)是高寒草地主要的磷素累积形态,其含量显著高于不施磷处理;(2)树脂交换态磷(Resin-Pi)、碳酸氢钠无机磷(NaHCO₃-Pi)、碳酸氢钠有机磷(NaHCO₃-Po)和氢氧化钠无机磷(NaOH-Pi)含量随施磷量增加整体呈先增加后降低趋势,表层土壤30 g/m²磷肥用量下其值均为最高,分别为21.54、22.94、65.86、64.48 mg/kg。酸溶性无机磷(HCl-Pi)随施磷量增加整体呈下...     

Biosorption of Cu(II) ions from synthetic and actual wastewater using three algal species

The use of three freshwater microalgal cultures—Chlorella sorokiniana, Anabaena laxa, and Hapalosiphon welwitschii—for sorption of copper(II) from synthetic Cu(II) solutions and Marinduque, Philippines, wastewater was studied. The optimum amount of biomass for the three species was 0.025 g dry weight. The optimum contact time for both C. sorokiniana and A. laxa was 1 h, whereas that of H. welwitschii was 30 min. All three species exhibited maximum Cu(II) sorption at pH 4.0–6.0. The Langmuir adsorption isotherm was the best fit model for the three species. The three cultures were found to be effective biosorbents when used in synthetic wastewaters of low concentration (10–30 ppm). Maximum Cu(II) reductions obtained were 88.2, 88.6, and 91.7% for the C. sorokiniana, A. laxa, and H. welwitschii cultures, respectively. C. sorokiniana, A. laxa, and H. welwitschii removed 5.70, 11.16, and 7.15% of Cu(II), respectively, when applied to wastewater taken from Consolidated Mines Inc. (CMI) containing around 150 ppm Cu(II). C. sorokiniana and A. laxa, in combination, exhibited 14.05% Cu(II) removal from CMI wastewater. Desorption with 0.11 M HCl effected 73.20, 64.54, and 70.85% removal of Cu(II) from the surfaces of C. sorokiniana, A. laxa, and H. welwitschii, respectively. SEM-EDS spectra of the three species confirmed the presence of Cu(II) on their surfaces. Presented at the 6th Meeting of the Asia Pacific Society of Applied Phycology, Manila, Philippines.     

Growth and nitrogenase activity of Azotobacter vinelandii in the presence of several phenolic acids

Growth and nitrogenase activity (acetylene reduction) of Azotobacter vinelandii in chemically defined N-free media were studied in the presence of p-hydroxybenzoic, vanillic, p-coumaric, and ferulic acids at concentrations from 0.01 to 1% (w/v). Growth and nitrogenase activity were only detected when the microorganism was cultured on p-hydroxybenzoic acid either as sole carbon source or mixed with other phenolic acids, suggesting that p-hydroxybenzoic acid could be utilized as a carbon source by A. vinelandii for growing under certain environmental conditions.     

Ammonia inhibition of nitrogenase activity in purple and green bacteria

Ammonia reversibly inhibits the nitrogenase activity not only in purple nonsulfur bacteria but in purple (Thiocapsa roseopersicina) and green (Chlorobium limicola forma thiosulfatophilum) sulfur bacteria as well.The complete inhibition of nitrogenase activity (acetylene reduction) is observed about 30 s after addition of NH ₄ ⁺ (2.5×10^-6 M) to cell suspensions. The pattern of ammonia inhibition of acetylene reduction in T. roseopersicina does not differ from the action of tetrabutylammonium and tetraphenylphosphonium (3 · 10^-6-5·10^-5 M) on nitrogenase activity of this bacterium.Simultaneously with the switch-off effect of NH ₄ ⁺ a considerable increase of ATP in cells of Rhodobacter sphaeroides and C. limicola f. thiosulphatophilum was observed.     

Nitrogenase activity of two genotypes of Acacia mangium as affected by phosphorus nutrition

The specific nodulation, nitrogenase activity (acetylene reduction) and budgets of carbon allocation to respiration by nodulated roots were examined in two provenances of Acacia mangium Willd. grown in a glasshouse for 17 weeks to investigate the effects of soil phosphorus and genotypes of the host plant on symbiotic nitrogen fixation. Application of phosphorus (0–80 mg P kg^-1 soil) increased specific nodulation (g nodule dry weight g^-1 plant dry weight) of provenance Ma11 by two-fold and the percentage of nodulated root respiration allocated to nitrogenase by 50%, but had no effect on specific activity of nitrogenase or specific respiration coupled with nitrogenase activity. Improved phosphorus nutrition increased the specific nitrogenase activity of provenance Ma9 by 2-fold, the percentage of nodulated root respiration allocated to nitrogenase, and specific nitrogenase-linked respiration by 50%, respectively, but had no effect on the specific nodulation. The percentage of respiration coupled with nitrogenase activity in nodulated root respiration by provenance Ma9 was 60–70% higher than that in provenance Ma11, regardless of phosphorus levels applied. At the optimal level of phosphorus addition (10 mg P kg^-1 soil), provenance Ma9 had a lower dry mass than provenance Ma11. This was accompanied by a lower nodulated root respiration and a higher percentage of nodulated root respiration allocated to nitrogenase activity in provenance Ma9.     

Effect of pesticides on growth,respiration and nitrogenase activity ofAzotobacter andAzospirillum

Pesticides (Brominal, Cuprosan and Fenvalerate) at 10 and 50 ppm suppressed growth, respiration and nitrogenase activity ofAzotobacter chroococcum, Azospirillum brasilense andAzospirillum lipoferum. The inhibitory effect on respiration ofAsm. lipoferum was most pronounced after 3 and 4 days.     

Short term effects of radiata pine and selected pasture species on soil organic phosphorus mineralisation

Silvopastoral systems comprise part of the continued expansion of conifer plantings on grassland in New Zealand. Greater understanding of the short term dynamics of soil organic P in such systems will further our knowledge about soil carbon and phosphorus relationships which will enable improved nutrient management in the field. A glasshouse experiment was carried out to examine the short-term effects (36 weeks) of combinations of radiata pine (Pinus radiata), lucerne (Medicago sativa L.) and perennial ryegrass (Lolium perenne L.) grown in the same soil type with a range of carbon (C) and phosphorus (P) levels on plant P uptake and the specific mineralisation rate (SMR). The SMR is defined as net mineralisation rate (i.e. gross mineralisation less microbial and geochemical uptake) and calculated from organic P decline as a percentage of organic P in the original soil before planting. This included an investigation of the effect of tree ectomycorrhizal (EM) hyphae on soil organic P. Plant P uptake was positively correlated with water soluble organic carbon (WSOC) and SMR, which in turn was closely related to soil C levels. The soils with high WSOC and C levels (which also contained high levels of labile inorganic and organic P) enabled high P uptake. Although P uptake was the greatest under radiata pine, the trees tended to deplete inorganic P to a lesser extent than the forages. When tree and forage species were combined, P uptake by forages was similar to when the forages were grown alone. The various soil and plant treatments significantly affected SMR. The two low C soils, showed the greatest organic P mineralisation while a high C soil, which contained significant levels of bicarbonate extracted inorganic P at planting and was under a long established undisturbed pasture, showed the least mineralisation. Trees grown alone showed the greatest SMR, EM hyphae and trees with lucerne were slightly lower than trees alone, while the forages showed the lowest SMR. The findings of this study showed that changes in organic P are strongly influenced by interactions between plant species (radiata pine, lucerne, ryegrass) and soil properties as determined by land use and management.     

Effect of nitrogen starvation on ammonium-inhibition of nitrogenase activity in Azotobacter chroococcum

When Azotobacter chroococcum cells grown in batch culture under N₂-fixing conditions were transferred to a medium lacking a nitrogen source, the cellular C/N ratio, the amount of alginic acid released into the external medium and the rate of endogenous respiration increased appreciably after 6 h to the exclusion of dinitrogen, whereas nitrogenase activity did not undergo any significant change. Nitrogen deficiency caused a decrease in the ammonium inhibition of nitrogenase activity from 95% inhibition at zero time to 14% after 6 h incubation under dinitrogen starvation, with no difference in the rate of ammonium utilization by N₂-fixing and N₂-starved cells being observed. This suggests that a balance of nitrogen and carbon assimilation is necessary for the ammonium inhibition of nitrogenase activity in A. chroococcum to take place.