O2 regulation and O2‐limitation of nitrogenase activity in root nodules of pea and lupin

The gas exchange characteristics of intact attached nodulated roots of pea (Pisum sativum cv. Finale X) and lupin (Lupinus albus cv. Ultra) were studied under a number of environmental conditions to determine whether or not the nodules regulate resistance to oxygen diffusion. Nitrogenase activity (H₂ evolution) in both species was inhibited by an increase in rhizosphere pO₂ from 20% to 30%, but recovered within 30 min without a significant increase in nodulated root respiration (CO₂ evolution). These data suggest that the nodules possess a variable barrier to O₂ diffusion. Also, nitrogenase activity in both species declined when the roots were either exposed to an atmosphere of Ar:O₂ or when the shoots of the plants were excised. These declines could be reversed by elevating rhizosphere pO₂, indicating that the inhibition of nitrogenase activity resulted from an increase in gas diffusion resistance and consequent O₂-limitation of nitrogenase-linked respiration. These results indicate that nodules of pea and lupin regulate their internal O₂ concentration in a manner similar to nodules of soybean, despite the distinct morphological and biochemical differences that exist between the nodules of the 3 species. Experiments in which total nitrogenase activity (TNA = H₂ production in Ar:O₂) in pea and lupin nodules was monitored while rhizosphere pO₂ was increased gradually to 100%, showed that the resistance of the nodules to O₂ diffusion maintains nitrogenase activity at about 80% of its potential activity (PNA) under normal atmospheric conditions. The O₂-limitation coefficient of nitrogenase (OLC_N= TNA/PNA) declined significantly with prolonged exposure to Ar:O₂ or with shoot excision. Together, these results indicate a significant degree of O₂-limitation of nitrogenase activity in pea and lupin nodules, and that yields may be increased by realizing full potential activity.     

N2 fixation by Acacia species increases under elevated atmospheric CO2

In the present study the effect of elevated CO₂ on growth and nitrogen fixation of seven Australian Acacia species was investigated. Two species from semi‐arid environments in central Australia (Acacia aneura and A. tetragonophylla) and five species from temperate south‐eastern Australia (Acacia irrorata, A. mearnsii, A. dealbata, A. implexa and A. melanoxylon) were grown for up to 148 d in controlled greenhouse conditions at either ambient (350 µmol mol^?1) or elevated (700 µmol mol^?1) CO₂ concentrations. After establishment of nodules, the plants were completely dependent on symbiotic nitrogen fixation. Six out of seven species had greater relative growth rates and lower whole plant nitrogen concentrations under elevated versus normal CO₂. Enhanced growth resulted in an increase in the amount of nitrogen fixed symbiotically for five of the species. In general, this was the consequence of lower whole‐plant nitrogen concentrations, which equate to a larger plant and greater nodule mass for a given amount of nitrogen. Since the average amount of nitrogen fixed per unit nodule mass was unaltered by atmospheric CO₂, more nitrogen could be fixed for a given amount of plant nitrogen. For three of the species, elevated CO₂ increased the rate of nitrogen fixation per unit nodule mass and time, but this was completely offset by a reduction in nodule mass per unit plant mass.     

The effect of excision on O2 diffusion and metabolism in soybean nodules

Nitrogen-fixing nodules of soybean [Glycine max (L.) Merr. cv. Maple Arrow inoculated with Bradyrhizobium japonicum USDA 16] were studied before and after excision from the root to determine the role the O₂ regulation plays in the inhibition of nodule activity and the potential for using excised nodules nodules in studies of nodule metabolism. Relative nitrogenase (EC 1.7.99.2) activity (H₂ evolution in N₂:O₂) and nodule respiration (CO₂ evolution) were monitored first in intact nodulated roots and then in freshly excised nodules of the same plant to determine the time course of the decline in nodule metabolism. Folowing excision, nitrogenase activity and respiration declined rapidly in the first minute and then more gradually. After 40 min the rate of H₂ evolution was only 14–28% of that in the intact plant. In some nodules activity declined steadily, and in others there was a partial recovery in activity ca 10 min after detachment. Infected cell O₂ concentration (O_i), measured by a spectro-photometric technique, also declined after nodule detachment with a time course similar to the declines in nitrogenase activity and respiration. Following excision, O_i levels declined rapidly from ca 21 nM in attached nodules to 8–12 nM at 4–10 min after excision and then more gradually to 2–3 nM O₂ at 30–40 min after excision. These results show that the nodules' permeability to gas diffusion continued to be regulated for up to 40 min after detachement. At 40 min after detachment, when excised nodules displayed steady-state rates of gas exchange, linear increases in pO₂ from 20 to 100% at 4% min^?1 resulted in recoveries of H² and CO² evolution, indicating that O_i limited nitrogenase activity durig this period, and that energy reserves were greatly in excess of the O₂ available for respiration. When detached nodules were equilibrated for 12 h at 20, 30 and 50% O₂, O_i values measured at supra-ambient pO₂ were greater than those at 20% O₂ and were linked with a more rapid decline in nitrogenase activity. Also, increases in external pO₂ (O_c) failed to stimulate nodule metabolism, suggesting that the nodules' energy reserves were no longer greatly in excess of their respiratory demands. It was concluded that soybean nodules could provide useful material for steady-state studies of nodule metabolism between 40 and 240 min after detachment, but to attain metabolic rates equivalent to in vivo rates the nodules must be exposed to above-ambient pO₂.     

Oxygen limitation of N2 fixation in stem-girdled and nitrate-treated soybean

The effects of increasing rhizosphere pO₂on nitrogenase activity and nodule resistance to O₂diffusion were investigated in soybean plants [Glycine max (L.) Merr. cv. Harosoy 63] in which nitrogenase (EC 1.7.99.2) activities were inhibited by (a) removal of the phloem tissue at the base of the stem (stem girdling), (b) exposure of roots to 10 mM NO₃over 5 days (NO₃-treated), or (c) partial inactivation of nitrogenase activity by an exposure of nodulated roots to 100 kPa O₂(O₂-inhibitcd). In control plants and in plants which had been treated with 100 kPa O₂, increasing rhizosphere O₂concentrations in 10 kPa increments from 20 to 70 kPa did not alter the steady-state nitrogenase activity. In contrast, in plants in which nitrogenase activities were depressed by stem girdling or by exposure to NO₃, increasing rhizosphere pO₂resulted in a recovery of 57 or 67%, respectively, of the initial, depressed rates of nitrogenase activity. This suggests that the nitrogenase activity of stem-girdled and NO₃-treated soybeans was O₂-limited. For each treatment, theoretical resistance values for O₂diffusion into nodules were estimated from measured rates of CO₂exchange, assuming a respiratory quotient of 1.1 and 0 kPa of O₂in the infected cells. At an external partial pressure of 20 kPa O₂, the stem-girdled and NO₃^--treated plants displayed resistance values which were 4 to 8.6 times higher than those in the nodules of the control plants. In control and O₂-inhibited plants, increases in pO₂from 20 to 70 kPa in 10 kPa increments resulted in a 2.5- to 3.9-fold increase in diffusion resistance to O₂, and had little effect on either respiration or nitrogenase activity. In contrast, in stem-girdled and NO₃^--treated plants, increases in external pO₂had little effect on diffusion resistance to O₂, but resulted in a 2.3- to 3.2-fold increase in nodule respiration and nitrogenase activity. These results are consistent with stem-girdling and NO₃^--inhibition treatments limiting phloem supply to nodules causing an increase in diffusion resistance to O₂at 20 kPa and an apparent insensitivity of diffusion resistance to increases in external pO₂.     

NO3-态氮对花生结瘤与固氮作用的影响

NO     

Laboratory incubations reveal potential responses of soil nitrogen cycling to changes in soil C and N availability in Mojave Desert soils exposed to elevated atmospheric CO2

Elevated atmospheric carbon dioxide (CO₂) has the potential to alter soil carbon (C) and nitrogen (N) cycling in arid ecosystems through changes in net primary productivity. However, an associated feedback exists because any sustained increases in plant productivity will depend upon the continued availability of soil N. We took soils from under the canopies of major shrubs, grasses, and plant interspaces in a Mojave Desert ecosystem exposed to elevated atmospheric CO₂ and incubated them in the laboratory with amendments of labile C and N to determine if elevated CO₂ altered the mechanistic controls of soil C and N on microbial N cycling. Net ammonification increased under shrubs exposed to elevated CO₂, while net nitrification decreased. Elevated CO₂ treatments exhibited greater fluxes of N₂O–N under Lycium spp., but not other microsites. The proportion of microbial/extractable organic N increased under shrubs exposed to elevated CO₂. Heterotrophic N₂‐fixation and C mineralization increased with C addition, while denitrification enzyme activity and N₂O–N fluxes increased when C and N were added in combination. Laboratory results demonstrated the potential for elevated CO₂ to affect soil N cycling under shrubs and supports the hypothesis that energy limited microbes may increase net inorganic N cycling rates as the amount of soil‐available C increases under elevated CO₂. The effect of CO₂ enrichment on N‐cycling processes is mediated by its effect on the plants, particularly shrubs. The potential for elevated atmospheric CO₂ to lead to accumulation of NH₄⁺ under shrubs and the subsequent volatilization of NH₃ may result in greater losses of N from this system, leading to changes in the form and amount of plant‐available inorganic N. This introduces the potential for a negative feedback mechanism that could act to constrain the degree to which plants can increase productivity in the face of elevated atmospheric CO₂.     

The relationship between nodule adenylates and the regulation of nitrogenase activity by O2 in soybean

Nodulated soybeans (Glycine max L. Merr, cv. Maple Arrow) were exposed to various physiological and environmental treatments to determine the relationship between nodule adenylate pools and the degree of O₂ limitation of nitrogenase. Adenylate energy charge (AEC = [ATP + 0.5 ADP]/[ATP + ADP + AMP]) and ATP/ADP ratios declined under conditions of decreased (10%) external pO₂ but increased in nodules exposed to elevated (30%) external pO₂. Nitrogenase activity was inhibited by both pO₂ treatments, but recovered towards initial levels within 45 min. AEC also returned to initial levels during this period. To account for these and related data in the literature, it was hypothesized that 1) legume nodules regulate infected cell O₂ concentration (Oi) to maintain adenylate pools at levels which limit respiratory metabolism: 2) treatments which decrease Oi alter the adenylate pools and further limit nodule metabolism; 3) treatments which increase Oi to levels in excess of a narrow range alter the adenylate pools and activate biochemical pathways which are not conducive to nitrogenase activity. In a preliminary test of these hypotheses, changes in AEC and ATP/ADP ratio were studied in nodules in which nitrogenase activity was inhibited by stem girdling, nitrate fertilization and exposure to an Ar:O₂ atmosphere. All three treatments caused an increased O₂ limitation of nodule respiration and nitrogenase activity. However, decreases in AEC were observed only in the stem girdling and nitrate fertilization treatment: Ar:O₂ exposure had no effect on whole nodule AEC. While this result challenged the hypotheses suggesting a central role for adenylates in the regulation of O₂-limited metabolism, it was noted that the Ar:O₂ treatment would differ from the other treatments in that it would have a specific effect on the ATP demands for NH₃ assimilation in the plant fraction. Since AEC and ATP/ADP ratio would be affected by both the rate of ATP synthesis (potentially an O₂-limited process) and the demand for ATP, changes in these parameters in the whole nodule may not be a reliable indicator of adenylate-mediated O₂ limitation. Futher studies are needed to examine in vivo changes in adenylate pools in the plant and bacteroid fractions in nodules which vary in their degree of O₂-limited metabolism.     

Utilization of the acetylene reduction assay to screen for tolerance of symbiotic N2 fixation to limiting P nutrition in common bean

The limitation of symbiotic nitrogen fixation due to P deficiency restricts the development of a sustainable agriculture, particularly in Mediterrancan and tropical soils. Common bean genotypes, APN18, BAT271, PVA846, POT51, G2633 and G12168, were grown in an aerated N-free nutrient solution at low (72 μmol plant^-1 week^-1) and control P supplies (360 μmol plant^-1 week^-1). Nitrogenase activity was estimated by in situ measurements of acetylene reduction activity (ARA) in a flow-through system. During the assays, maximum values of ARA (peak ARA) were reached between 20 and 30 min after exposure to C₂H₂, depending on P treatment and growth stage. Thereafter, a decline in C₂H₄ evolution was observed. This decline was most pronounced in low-P plants and there was a significant genotypic effect. ARA per plant was decreased by P deficiency, mostly because nodulation was delayed and the number and mass of nodules were reduced. The ARA decrease during pod filling was also activated by P deficiency. ARA per g dry weight nodule was increased by P deficiency in G2633 and G12168, unchanged in APN18, BAT271 and POT51 and decreased in PVA846. Except for the climbing type IV G2633, total N at harvest for all P treatments was correlated with the cumulative value of peak ARA and with peak ARA at early pod-filling which was the highest peak ARA throughout the growth cycle of type III bushy genotypes. We conclude that if phenology and growth habit are carefully considered, peak ARA is a reliable screen of genotypes for N² fixation tolerance to P deficiency. Selection of lines with early nodulation under P deficiency is also advisable, and the effect of P deficiency on the nodule functioning needs to be considered.     

Carbon and nitrogen partitioning in young nodulated pea (wild type and nitrate reductase-deficient mutant) plants exposed to NH4NO3

Carbon and nitrogen partitioning was examined in a wild-type and a nitrate reductase-deficient mutant (A317) of Pisum sativum L. (ev. Juneau), effectively inoculated with two strains of Rhizobium leguminosarum (128C23 and 128C54) and grown hydroponically in medium without nitrogen for 21 days, followed by a further 7 days in medium without and with 5 mM NH₄NO₃. In wild-type symbioses the application of NH₄NO₃ significantly reduced nodule growth, nitrogenase (EC 1.7.99.2) activity, nodule carbohydrates (soluble sugars and starch) and allocation of [¹⁴C]-labelled (NO₃⁻, NH₄⁺, amino acids) in roots. In nodules, there was a decline in amino acids together with an increase in inorganic nitrogen concentration. In contrast, symbioses involving A317 exhibited no change in nitrogenase activity or nodule carbohydrates, and the concentrations of all nitrogenous solutes measured (including asparagine) in roots and nodules were enhanced. Photosynthate allocation to the nodule was reduced in the 128C23 symbiosis. Nitrite accumulation was not detected in any case. These data cannot be wholly explained by either the carbohydrate deprivation hypothesis or the nitrite hypothesis for the inhibition of symbiotic nitrogen fixation by combined nitrogen. Our result with A317 also provided evidence against the hypothesis that NO₃⁻ and NH₄⁺ or its assimilation products exert a direct effect on nitrogenase activity. It is concluded that more than one legume host and Rhizobium strain must be studied before generalizations about Rhizobium /legume interactions are made.     

HETEROCYST DEVELOPMENT AND LOCALIZATION OF CYANOPHYCIN IN N2-FIXING CULTURES OF ANABAENA SP. PCC 7120 (CYANOBACTERIA)

The process of N₂ fixation in the filamentous cyanobacterium Anabaena sp. PCC 7120 is known to occur in terminally differentiated cells called heterocysts. This study is concerned with a morphological and immunocytochemical analysis of the developing heterocysts. The heterocysts continue a developmental process after synthesis of the specialized cell wall and the formation of the proheterocyst. The initial stages were described by Wilcox et al. (1973) and designated stages 1 through 7, with stages 5–7 associated with the maturing heterocyst. We now designate a stage 8 as the postmaturation stage, based on physiological and ultrastructural evidence. Immunocytochemistry to detect the nitrogenase protein NifH and the nonribosomally synthesized polypeptide cyanophycin demonstrated a complementary accumulation of these polypeptides. Accumulation of the nitrogenase protein was greatest at stages 5 and 6 and then declined precipitously. Cyanophycin was more prevalent after late stage 6 and was primarily associated with the polar nodule (polar plug) and the neck connecting the heterocyst with the adjoining vegetative cell. We suggest that the cyanophycin-containing polar plug is a key intermediate in the storage of fixed nitrogen in the heterocyst, a result consistent with the suggestion first made by Carr (1988) that cyanophycin exists as a dynamic reservoir of fixed nitrogen within the heterocysts.     

Oxygen-induced recovery from short-term nitrate inhibition of N2 fixation in white clover plants from spaced and dense stands

Nitrogenase (N₂ase; EC 1.18.6.1) activity (H₂ evolution) and root respiration (CO₂ evolution) were measured under either N₂:O₂ or Ar:O₂ gas mixtures in intact nodulated roots from white clover ( Trifolium repens L.) plants grown either as spaced or as dense stands. The short-term nitrate (5 m M ) inhibition of N₂-fixation was promoted by competition for light between clover shoots, which reduced CO₂ net assimilation rate. Oxygen-diffusion permeability of the nodule declined during nitrate treatment but after nitrate removal from the liquid medium its recovery parallelled that of nitrogenase activity. Rhizosphere pO₂ was increased from 20 to 80 kPa under N₂:O₂. A simple mono-exponential model, fitted to the nodule permeability response to pO₂, indicated NO⁻₃ induced changes in minimum and maximum nodule O₂-diffusion permeability. Peak H₂ production rates at 80 kPa O₂ and in Ar:O₂ were close to the pre-decline rates at 20 kPa O₂. At the end of the nitrate treatment, this O₂-induced recovery in nitrogenase activity reached 71 and 82%; for clover plants from spaced and dense stands, respectively. The respective roles of oxygen diffusion and phloem supply for the short-term inhibition of nitrogenase activity in nitrate-treated clovers are discussed.     

New preparations and molecular structures of cis-MCl2(Ph2PCH2PPh2)2, M = Ru,Os and trans-RuCl2(Ph2PCH2PPh2)2

The title compounds were made by reacting bis(diphenylphosphino)methane (dppm) with reduced solutions of OsCl₆^4? and Ru₂OCl₁₀^4?. The crystal and molecular structures of these compounds have been determined form three-dimensional X-ray study. The cis-isomers crystallize with one CHCl₃ per molecule of the complex. All three compounds crystallize in the monoclinic space group P2₁/n with unit cell dimensions as follows: Cis-OsCl₂(dppm)₂·CHCl₃: a = 13.415(4) Å, b = 22.859(4) Å, c = 16.693(3) Å, β = 105.77(3)°, V = 4926(3) ų, Z = 4. cis-RuCl₂(dppm)₂·CHCl₃: a = 13.442(3) Å, b = 22.833(7) Å, c = 16.750(4) Å, β = 105.53(2)°, V = 4953(3) ų, Z = 4. trans-RuCl₂(dppm)₂: a = 11.368(7) Å, b = 10.656(6) Å, c = 18.832(12) Å; β = 103.90(6)°, V = 2213(7) ų; Z = 2. The structures were refined to R = 0.044 (R_w = 0.055) for cis-OsCl₂(dppm)₂·CHCl₃; R = 0.065 (R_w = 0.079) for cis-RuCl₂(dppm)₂·CHCl₃ and R = 0.028 (R_w = 0.038) for trans-RuCl₂(dppm)₂. The complexes are six coordinate with stable four-membered chelate rings. The PMP angle in the chelate rings is ca. 71° in each case.     

Crystal structures of MoCl2(O)(O2)(OPMePh2)2 and mixtures of MoCl2(O2)(OPPh3)2 and MoCl2(O)(O2)(OPPh3)2: allylic alcohol isomerization studies using MoCl2(O)(O2)(OPMePh2)2

Adding one equivalent of H₂O₂ to compounds of stoichiometry MoCl₂(O)₂(OPR₃)₂, OPR₃ = OPMePh₂ or OPPh₃, leads to the formation of oxo-peroxo compounds MoCl₂(O)(O₂)(OPR₃)₂. The compound MoCl₂(O)(O₂)(OPMePh₂)₂ crystallized with an unequal disorder, 63%:37%, between the oxo and peroxo ligands, as verified by single-crystal X-ray diffractometry, and can be isolated in reasonable yields. MoCl₂(O)(O₂)(OPPh₃)₂, was not isolated in pure form, co-crystallized with MoCl₂(O)₂(OPPh₃)₂ in two ratios, 18%:82% and 12%:88%, respectively, and did not contain any disorder in the arrangement of the oxo and peroxo groups. These complexes accomplish the isomerization of various allylic alcohols. A mechanism of this reaction has been constructed based on ¹⁸O isotopic studies and involves exchange between the alcohol and metal bonded O atoms.     

Achiral internucleoside linkages: CH2-CH2-NH and nH-CH2-CH2 linkages

The synthesis of CH₂-CH₂-NH and NH-CH₂-CH₂ internucleoside linkages are described. Antisense oligonucleosides containing these dimer modifications hybridized to the sense sequence. Furthermore incorporation of these backbone modifications enhanced the nuclease resistance of the antisense strand.     

Selective synthesis of triangular cluster oxido-sulfidocomplexes of Mo and W: High yield preparations of [Mo3O2S2(H2O)9], [W3O2S2(H2O)9], [W2MoO2S2(H2O)9] and their derivatization

Reaction of [Mo₂O₂(μ-S)₂(H₂O)₆]²⁺ with Mo(CO)₆ or metallic Mo under hydrothermal conditions (140 °C, 4 M HCl) gives oxido-sulfido cluster aqua complex [Mo₃(μ₃-S)(μ-O)₂(μ-S)(H₂O)₉]⁴⁺ (1). Similarly, [W₃(μ₃-S)(μ-O)₂(μ-S)(H₂O)₉]⁴⁺ (2) is obtained from [W₂O₂(μ-S)₂(H₂O)₆]²⁺ and W(CO)₆. While reaction of [Mo₂O₂(μ-S)₂(H₂O)₆]²⁺ with W(CO)₆ mainly proceeds as simple reduction to give 1, [W₂O₂(μ-S)₂(H₂O)₆]²⁺ with Mo(CO)₆ produces new mixed-metal cluster [W₂Mo(μ₃-S)(μ-O)₂(μ-S)(H₂O)₉]⁴⁺ (3) as main product. From solutions of 1 in HCl supramolecular adduct with cucurbit[6]uril (CB[6]) {[Mo₃O₂S₂(H₂O)₆Cl₃]₂CB[6]}Cl₂⋅18H₂O (4) was isolated and structurally characterized. The aqua complexes were converted into acetylacetonates [M₃O₂S₂(acac)₃(py)₃]PF₆ (M₃ = Mo₃, W₃, W₂Mo; 5a-c), which were characterized by X-ray single crystal analysis, electrospray ionization mass spectrometry and ¹H NMR spectroscopy. Crystal structure of (H₅O₂)(Me₄N)₄[W₃(μ₃-S)(μ₂-S)(μ₂-O)₂(NCS)₉] (6), obtained from 2, is also reported.     

Solution-mediated syntheses and characterizations of two new zincophosphites [C6H14N2]0.5[Zn(H2PO3)2] and [C4H12N2]0.5[(CH3)2NH2][Zn2(HPO3)3]

Two new zincophosphites [C₆H₁₄N₂]_0.5[Zn(H₂PO₃)₂] 1 and [C₄H₁₂N₂]_0.5[(CH₃)₂NH₂][Zn₂(HPO₃)₃] 2 have been solvothermally synthesized in mixed solvents of N,N-dimethylformamide (DMF) and 1,4-dioxane (DOA), respectively. Single-crystal X-ray diffraction analysis reveals that compound 1 exhibits a neutral inorganic chain formed by ZnO₄ and HPO₂(OH) units. Interestingly, the left- and right-handed hydrogen-bonded helical chains are alternately formed via the hydrogen-bonds between two adjacent chains. Compound 2 exhibits a layer structure with 4- and 12-MRs formed by ZnO₄ and HPO₃ units, in which two kinds of organic amine molecules both act as countercations to compensate the overall negative electrostatic charge of the anionic network.