15N2 measurement of nitrogen fixation by legumes and actinorhizals: theory and practice

A gas-tight chamber has been constructed to calibrate the ¹⁵N isotope dilution method against direct ¹⁵N₂ measurements. The theoretical basis for such estimates is given, and the practical problems associated with the experiments are discussed.     

Adaptation of methods for evaluating N2 fixation in food legumes and legume cover crops

Methods for partitioning the nitrogen assimilated by nodulated legumes, between nitrogen derived from soil sources and from N₂ fixation, are described as applied in peninsular Malaysia. The analysis of nitrogenous components translocated from the roots to the shoots of nodulated plants in the xylem sap is outlined, with some precautions to be observed for applications in the tropics. Some examples of the use of the technique in surverying apparent N₂ fixation by tropical legumes, in studying interrow cropping in plantation systems and in assessing effects of experimental treatments on N₂ fixation by food legumes, are described. Techniques for assesing N₂ fixation by means of¹⁵N abundance have been used to show that applications of nitrogenous fertilizers commonly used in Malaysia for soybeans depress N₂ fixation, that similar results are obtained with natural abundance and¹⁵N-enrichment methods and that, in at least two locations in Malaysia, differences between the natural abundance of¹⁵N in plant-available soil nitrogen and in atmospheric N₂ are great enough to permit application to measurement of N₂ fixation by leguminous crops.     

The essentiality of calcium ion in the enzymatic activity of Taiwan cobra phospholipase A2

In order to address the mechanism whereby Ca²⁺ wad crucial for the manifestation of the enzymatic activity of phospholipase A₂ (PLA₂), four divalent cations were used to assess their influences on the catalytic activity and the fine structures ofNaja naja atra PLA₂. It was found that substitution of Mg²⁺ or Sr²⁺ for Ca²⁺ in the substrate solution caused a decrease in the PLA₂ activity to 77.5% or 54.5%, respectively, of that in the presence of Ca²⁺. However, no PLA₂ activity was observed with the addition of Ba²⁺. With the exception of Mg²⁺, the nonpolarity of the 8-anilinonaphthalene-1-sulfonate (ANS)-binding site of PLA₂ markedly increased with the binding of cations to PLA₂. In the meantime, the accessibilities of Lys-6 (65) and Tyr-3 (63) toward trinitrobenzene sulfonate andp-nitrobenzenesulfonyl fluoride were enhanced by the addition of Ca²⁺, Sr²⁺, and Ba²⁺, but not by Mg²⁺. The order of the ability of cations to enhance the ANS fluorescence and the reactivity of Lys and Tyr residues toward modified reagents was Ba²⁺> Sr²⁺> Ca²⁺> Mg²⁺, which was the same order as the increase in their atomic radii. These results, together with the observations that the ANS molecule binds at the active site of PLA₂ and that Tyr-3, Lys-6, and Tyr-63 of PLA₂ are involved in the binding with the substrate, suggest that the binding of Ca²⁺ to PLA₂ induces conformational changes at the active site and substrate-binding site. However, the smaller atomic radius with Mg²⁺ or the bigger atomic radii with Sr²⁺ and Ba²⁺ might render the conformation improperly rearranged after their binding to PLA₂ molecule.     

Symbiotic N2 fixation by soybean in organic and conventional cropping systems estimated by 15N dilution and 15N natural abundance

Nitrogen (N) is often the most limiting nutrient in organic cropping systems. N₂ fixing crops present an important option to improve N supply and to maintain soil fertility. In a field experiment, we investigated whether the lower N fertilization level and higher soil microbial activity in organic than conventional systems affected symbiotic N₂ fixation by soybean (Glycine max, var. Maple Arrow) growing in 2004 in plots that were since 1978 under the following systems: bio-dynamic (DYN); bio-organic (ORG); conventional with organic and mineral fertilizers (CON); CON with exclusively mineral fertilizers (MIN); non-fertilized control (NON). We estimated the percentage of legume N derived from the atmosphere (%Ndfa) by the natural abundance (NA) method. For ORG and MIN we additionally applied the enriched ¹⁵N isotope dilution method (ID) based on residual mineral and organic ¹⁵N labeled fertilizers that were applied in 2003 in microplots installed in ORG and MIN plots. These different enrichment treatments resulted in equal %Ndfa values. The %Ndfa obtained by NA for ORG and MIN was confirmed by the ID method, with similar variation. However, as plant growth was restricted by the microplot frames the NA technique provided more accurate estimates of the quantities of symbiotically fixed N₂ (Nfix). At maturity of soybean the %Ndfa ranged from 24 to 54%. It decreased in the order ORG > CON > DYN > NON > MIN, with significantly lowest value for MIN. Corresponding Nfix in above ground plant material ranged from 15 to 26 g N m^-2, with a decreasing trend in the order DYN = ORG > CON > MIN > NON. For all treatments, the N withdrawal by harvested grains was greater than Nfix. This shows that at the low to medium %Ndfa, soybeans did not improve the N supply to any system but removed significant amounts of soil N. High-soil N mineralization and/or low-soil P availability may have limited symbiotic N₂ fixation.     

Short-term effects of a dung pat on N2 fixation and total N uptake in a perennial ryegrass/white clover mixture

The short-term effects of a simulated cattle dung pat on N₂ fixation and total uptake of N in a perennial ryegrass/white clover mixture was studied in a container experiment using sheep faeces mixed with water to a DM content of 13%. We used a new ¹⁵N cross-labelling technique to determine the influence of dung-pat N on N₂ fixation in a grass/clover mixture and the uptake of dung N in grass and clover. The proportion of N in clover derived from N₂ fixation (%Ndfa) varied between 88–99% during the 16 weeks following application of the dung. There was no effect of dung on the %Ndfa in clover grown in mixture, whereas the %Ndfa in clover grown in pure stand decreased (nominal 2–3%) after dung application. Dung did not influence the amount of N₂ fixed, and the uptake of dung N in grass and clover proceeded at an almost constant rate. After 16 weeks, 10% of the applied dung N was taken up by grass and clover, 57% had been incorporated in the soil by faunal activity and 27% remained in residual dung on the soil surface. The dung N unaccounted for (7%) was probably lost by ammonia volatilisation and denitrification. The uptake of dung N in grass/clover mixtures in the field was similarly followed by using simulated ¹⁵N-labelled dung pats. The total dry matter production and N yields increased in the 0–30 cm distance from the edge of the dung patch, but the proportion of clover decreased. Thirteen months after application of the dung 4% of the applied dung N was recovered in the harvested herbage, 78% was recovered from the soil and the residual dung, and 18% was not accounted for. It is concluded that N₂ fixation in the dung patch border area in grass/clover mixtures is not influenced directly by the release of N from dung pats in the short term. However the amount of N₂ fixed may be reduced, if the growth of clover is reduced in the patch border area.     

Estimation of symbiotically fixed nitrogen in field grown soybeans: An application of natural15N/14N abundance and a low level15N-tracer technique

Summary Plants from agricultural and natural upland ecosystem were investigated for¹⁵N content to evaluate the role of symbiotic N₂-fixation in the nitrogen nutrition of soybean. Increased yields and lower δ¹⁵N values of nodulating soybeansvs, non-nodulating isolines gave semi-quantitative estimates of N₂ fixation. A fairly large discrepancy was found between estimations by δ¹⁵N and by N yield at 0 kg N/ha of fertilizer. More precise estimates were made by following changes in plant δ¹⁵N when fertilizer δ¹⁵N was varied near¹⁵N natural abundance level. Clearcut linear relationships between δ¹⁵N values of whole plants and of fertilizer were obtained at 30 kg N/ha of fertilizer for three kinds of soils. In experimental field plots, nodulating soybeans obtained 13±1% of their nitrogen from fertilizer, 66±8% from N₂ fixation and 21±10% from soil nitrogen in Andosol brown soil; 30%, 16% and 54% in Andosol black soil; 7%, 77% and 16% in Alluvial soil, respectively. These values for N₂ fixation coincided with each corresponding estimation by N yield method. Other results include: 1)¹⁵N content in upland soils and plants was variable, and may reflect differences in the mode of mineralization of soil organics, and 2) nitrogen isotopic discrimination during fertilizer uptake (δ¹⁵N of plant minus fertilizer) ranged from −2.2 to +4.9‰ at 0–30 kg N/ha of fertilizer, depending on soil type and plant species. The proposed method can accurately and relatively simply establish the importance of symbiotic nitrogen fixation for soybeans growing in agricultural settings.     

N2 fixation and nitrogen allocation to above and below ground plant parts in red clover-grasslands

Leys, used for grazing or production of forage to be conserved as silage or hay, are very important crops in northern areas. In order to measure the N₂ fixation in leys of varying ages and during different parts of the season, detailed measurements were taken of yield, N₂ fixation and the amounts of N remaining in the field after harvesting red clover (Trifolium pratense L.)-grass leys at a site in northern Sweden, where they are generally harvested twice per growing season. Entire plants, including stubble and roots, were sampled at the time of first and second harvest and, in addition, at the end of the growing season in three neighbouring fields, carrying a first, a second and a third year ley, respectively. N₂ fixation was measured by both ¹⁵N isotope dilution (ID) and ¹⁵N natural abundance (NA) methods. The proportion of clover dry matter (DM) in the stands increased from the first to the second harvest, but the grasses dominated throughout the entire season, especially below ground. The N concentrations, in both herbage and whole plants, were about twice as high in the clover as in the grasses. Seasonal variations in N concentrations were minor, and total N contents followed the same trends as DM. The clover acquired nearly all of its N from N₂ fixation: the proportion of N in clover herbage derived from N₂ fixation was often >0.8 throughout the season. The variations in the amounts of N₂ fixed during the course of the season corresponded well to the seasonal changes in clover biomass. Amounts of fixed N₂ allocated to clover herbage during the whole season were in the range 4 to 6 g N m⁻² in this unusually rainy year. Calculations of daily N allocation rates to herbage showed that N uptake rates were similar, and high, in grasses during May–June and July–August, while N₂ fixation rates in clover were about 10-fold as high in July–August as in May–June, reflecting the need for N in clover growth. The proportion of N remaining in clover stubble and roots after the first and second harvests was about 60 and 25%, respectively, while about 60% of the N in grasses remained in stubble and roots after both harvests. The considerable amounts of biomass and N that were left in field after harvesting red clover-grass leys are important for re-growth of the plants and provide substantial N fertilization for the next crop in the crop rotation.     

The15N methodology in estimating N2 fixation by vetch and pea grown in pure stand or in mixtures with oat

Cereal-legume mixtures are frequently the best management decision for forage production instead of growing crops in pure stands. Nitrogen fertilization of cereal-legume mixtures is questionable since combined nitrogen could depress N₂ fixation by legumes. The objectives of this study were (1) to examine the effect of N fertilization on N₂ fixation by vetch and field peas in pure and in mixed stands with oats, and (2) to examine if there is any transfer of N from legumes to associated cereals. The field experiment was conducted for two growing seasons. The treatments were pure stands of vetch, pea and oats, and the mixtures of the two legumes with oats at the seeding ratios 90:10 and 75:25, fertilized with labelled¹⁵N at the rates of 15 and 90 kg N ha⁻¹. Nitrogen fertilization of 90 kg N ha⁻¹ suppressed N₂ fixation in both legumes grown in pure and in mixed stands. Crops grown in mixtures in many instances had lower atom %¹⁵N excess. Whether this was due to high N₂ fixation in the case of legume and transfer in the case of oat or the differences were due to practical problems of the¹⁵N technique is not clearly shown by the results, so based on the literature the aspect is discussed as well as the precautions which should be considered in using the¹⁵N technique in such studies.     

Manganese(III) complexes of N2O2 donor 5-bromosalicylideneimine ligands: Combined effects of electron withdrawing substituents and chelate ring size variations on electrochemical properties

A series of mononuclear manganese(III) complexes of formulae [Mn(L)(X)(H₂O)] (1-13) and [Mn(L)(X)] (14-17) (X = ClO₄, F, Cl, Br, I, NCS, N₃), derived from the Schiff bases of 5-bromosalicylaldehyde and different types of diamine (1,2-diaminoethane, 1,2-diaminopropane, 1,3-diaminopropane and 1,4-diaminobutane), have been synthesized and characterized by the combination of IR, UV-Vis spectroscopies, cyclic voltammetry and by X-ray crystallography. The redox properties of all the manganese(III) complexes show grossly identical features consisting of a reversible or quasireversible Mn^III/Mn^II reduction. Besides Mn^III/Mn^II reduction, the complexes 4, 5, 10, 13 and 16 also show reversible or quasireversible Mn^III/Mn^IV oxidation. A linear correlation has been found for the complexes 5, 7, 11 and 13 [Mn(L²)(X)(H₂O)] (X = F, Cl, Br, I) when E_1/2 [Mn^III/Mn^II] is plotted against Mulliken electronegativities (χ_M). The effect of the flexibility of the ligand on redox potential has been studied. It has been observed that the manganese(II) state is stabilized with increasing flexibility of the ligand environment. The crystal structure of 6 shows an octahedral geometry.     

Synthesis and characterization of three group 10 complexes containing nido-carborane diphosphine: [MCl(PPh3){7,8-(PPh2)2-7,8-C2B9H10}] (M = Ni, Pd, Pt)

Three group 10 complexes containing nido-carborane diphosphine, [NiCl(PPh₃){7,8-(PPh₂)₂-7,8-C₂B₉H₁₀}] (1), [PdCl(PPh₃){7,8-(PPh₂)₂-7,8-C₂B₉H₁₀}] · 1.25CH₂Cl₂ (2) and [PtCl(PPh₃){7,8-(PPh₂)₂-7,8-C₂B₉H₁₀}] · 2.5CH₂Cl₂ (3) have been synthesized by the reactions of [M(PPh₃)₂Cl₂] (M = Ni, Pd, Pt) with closo carborane diphosphine 1,2-(PPh₂)₂-1,2-C₂B₁₀H₁₀ in ethanol. For complex 3, it could also be obtained under solvothermal condition. All three complexes were characterized by elemental analysis, FT-IR, ¹H and ¹³C NMR spectroscopy and X-ray structure determination. Single crystal structures show that their structures are similar to each other. In each complex, the nido [7,8-(PPh₂)₂-7,8-C₂B₉H₁₀]⁻, which resulted from the degradation of the initial closo ligand 1,2-(PPh₂)₂-1,2-C₂B₁₀H₁₀ during the reaction process, was coordinated bidentately through the P atoms to M(II) ion, and this resulted in a stable five-membered chelating ring between the bis-diphosphine ligand and the metal. The coordination mode of the metal can be described as a slightly distorted square-planar, in which the remaining two positions were occupied by one Cl⁻ and one PPh₃ group.     

Determination of muscle protein synthesis rates in fish using H2O and H NMR analysis of alanine

Following administration of deuterated water (²H₂O), the fractional synthetic rate (FSR) of a given endogenous protein can be estimated by ²H-enrichment quantification of its alanine residues. Currently, this is measured by mass spectrometry following a derivatization procedure. Muscle FSR was measured by ¹H/²H NMR analysis of alanine from seabass kept for 6 days in 5% ²H-enriched saltwater, following acid hydrolysis and amino acid isolation by cation-exchange chromatography of muscle tissue. The analysis is simple and robust, and provides precise measurements of excess alanine ²H-enrichment in the 0.1–0.4% range from 50 mmol of alanine recovered from muscle protein.     

Proton NMR resonance assignments and surface accessibility of tryptophan residues of a dimeric phospholipase A2 from Trimeresurus flavoviridis

Proton NMR spectra of a dimeric phospholipase A₂ from Trimeresurus flavoviridis have been recorded. N-1 proton resonances of the tryptophan indole rings have been detected and assigned to specific positions, Trp-3/Trp-30, Trp-68 and Trp-108, by comparing the spectra of the enzyme derivatives with tryptophans oxidized to differing extents. Photo-CIDNP experiments have revealed that Trp-68 and Trp-108 are exposed while Trp-3 and Trp-30 are buried in the molecule. This is consistent with the X-ray crystal structure of a homologous phospholipase A₂ from Crotalus atrox where residues 3 and 30 are located at a dimer interface, but inconsistent with the results of stepwise oxidation of tryptophan residues.     

Contribution of N2 fixing cyanobacteria to rice production: availability of nitrogen from 15N-labelled cyanobacteria and ammonium sulphate to rice

This study investigate the potential contribution of nitrogen fixation by indigenous cyanobacteria to rice production in the rice fields of Valencia (Spain). N₂-fixing cyanobacteria abundance and N₂ fixation decreased with increasing amounts of fertilizers. Grain yield increased with increasing amounts of fertilizers up to 70 kg N ha^-1. No further increase was observed with 140 kg N ha^-1. Soil N was the main source of N for rice, only 8–14% of the total N incorporated by plants derived from ¹⁵N fertilizer. Recovery of applied ¹⁵N-ammonium sulphate by the soil–plant system was lower than 50%. Losses were attributed to ammonia volatilization, since only 0.3–1% of applied N was lost by denitrification. Recovery of ¹⁵N from labeled cyanobacteria by the soil–plant system was higher than that from chemical fertilizers. Cyanobacterial N was available to rice plant even at the tillering stage, 20 days after N application. This revised version was published online in June 2006 with corrections to the Cover Date.     

Elevated CO2 and nutrient addition after soil N cycling and N trace gas fluxes with early season wet-up in a California annual grassland

We examined the effects of growth carbon dioxide (CO₂)concentration and soil nutrient availability on nitrogen (N)transformations and N trace gas fluxes in California grasslandmicrocosms during early-season wet-up, a time when rates of Ntransformation and N trace gas flux are high. After plant senescenceand summer drought, we simulated the first fall rains and examined Ncycling. Growth at elevated CO₂ increased root productionand root carbon:nitrogen ratio. Under nutrient enrichment, elevatedCO₂ increased microbial N immobilization during wet-up,leading to a 43% reduction in gross nitrification anda 55% reduction in NO emission from soil. ElevatedCO₂ increased microbial N immobilization at ambientnutrients, but did not alter nitrification or NO emission. ElevatedCO₂ did not alter soil emission of N₂O ateither nutrient level. Addition of NPK fertilizer (1:1:1) stimulatedN mineralization and nitrification, leading to increased N₂Oand NO emission from soil. The results of our study support a mechanisticmodel in which elevated CO₂ alters soil N cycling and NOemission: increased root production and increased C:N ratio in elevatedCO₂ stimulate N immobilization, thereby decreasingnitrification and associated NO emission when nutrients are abundant.This model is consistent with our basic understanding of how C availabilityinfluences soil N cycling and thus may apply to many terrestrial ecosystems.     

Effects of planted tree fallows on soil nitrogen dynamics,above-ground and root biomass,N2-fixation and subsequent maize crop productivity in Kenya

The effects of planted fallows of Sesbania sesban (L.) Merr. and Calliandra calothyrsus (Meissner) on soil inorganic nitrogen dynamics and two subsequent maize crops were evaluated under field conditions in the highlands of eastern Kenya. Continuous unfertilised maize, maize/bean rotation and natural regrowth of vegetation (weed fallow) were used as control treatments. The proportion of symbiotic N₂-fixation was estimated by measuring both leaf ¹⁵N enrichment and whole-plant ¹⁵N enrichment by the ¹⁵N dilution technique for Sesbania and Calliandra, using Eucalyptus saligna (Sm.) and Grevillea robusta (A. Cunn) as reference species. Above- and below-ground biomass and N contents were examined in Sesbania, Calliandra, Eucalyptus and Grevillea 22 months after planting. Both the content of inorganic N in the topsoil and the quantity of N mineralised during rainy seasons were higher after the Sesbania fallows than after the other treatments. Compared to the continuous unfertilised maize treatment, both residual crop yields were significantly higher when mineral N (one application of 60 kg N ha^–1) was added. Furthermore, the second crop following the Sesbania fallow was significantly higher than the continuous maize crop. The above-ground biomass of the trees at final harvest were 31.5, 24.5, 32.5 and 43.5 Mg ha^–1 for the Sesbania, Calliandra, Grevillea and Eucalyptus, respectively. For the total below-ground biomass the values for these same tree species were 11.1, 15.5, 17.7, and 19.1 Mg ha^–1, respectively, of which coarse roots (>2 mm), including tap roots, amounted to 70–90%. About 70–90% of the N in Sesbania, and 50–70% in Calliandra, was derived from N₂-fixation. Estimates based on leaf ¹⁵N enrichment and whole-plant ¹⁵N enrichment were strongly correlated. The N added by N₂-fixation amounted to 280–360 kg N ha^–1 for Sesbania and 120–170 kg N ha^–1 for Calliandra, resulting in a positive N balance after two maize cropping seasons of 170–250 kg N ha^–1 and 90–140 kg N ha^–1, for Sesbania and Calliandra, respectively. All the other treatments gave negative N balances after two cropping seasons. We conclude that Sesbania sesban is a tree species well suited for short duration fallows due to its fast growth, high nutrient content, high litter quality and its ability to fix large amounts of N₂ from the atmosphere.     

Symbiotic N2-fixation in alpine tundra: ecosystem input and variation in fixation rates among communities

Annual inputs of symbiotic N₂-fixation associated with 3 species of alpine Trifolium were estimated in four alpine communities differing in resource supplies. We hypothesized that fixation rates would vary according to the degree of N, P, and water limitation of production, with the higher rates of fixation in N limited communities (dry meadow, moist meadow) and lower rates in P and water limited communities (wet meadow, fellfield). To estimate N₂-fixation rates, natural abundance of N isotopes (¹⁵N) were measured in field collected Trifolium and reference plants and in Trifolium plants grown in N-free medium in a growth chamber. All three Trifolium species relied on a large proportion of atmospherically-fixed N₂ to meet their N requirements, ranging from 70 to 100%. There were no apparent differences in the proportion of plant N derived from fixation among the communities, but differences in the contribution of the Trifolium species to community cover resulted in a wide range of annual N inputs from fixation, from 127 mg m^–2 year^–1 in wet meadows to 810 mg m^–2 year^–1 in fellfields. Annual spatially integrated input of symbiotic N₂-fixation to Niwot Ridge, Colorado was estimated at 490 mg m^–2 year^–1 (5 kg ha^–1 year^–1), which is relatively high in the context of estimates of net N mineralization and N deposition.     

A field method of determining NH 4 + and NO 3 - uptake kinetics in intact roots: Effects of CO2 enrichment on trees and crop species

Models describing plant and ecosystem N cycles require an accurate assessment of root physiological uptake capacity for NH ₄ ⁺ and NO ₃ ^- under field conditions. Traditionally, rates of ion uptake in field-grown plants are determined by using excised root segments incubated for a short period in an assay solution containing N either as a radioactive or stable isotope tracer (e.g., ³⁶ClO₃ as a NH ₄ ⁺ analogue, ¹⁴CH₃NH₃ as an NO ₃ ^- analogue or ¹⁵NH ₄ ⁺ and ¹⁵NO ₃ ^- ). Although reliable, this method has several drawbacks. For example, in addition to radioactive safety issues, purchase and analysis of radioactive and stable isotopes is relatively expensive and can be a major limitation. More importantly, because excision effectively interrupts exchange of compounds between root and shoot (e.g., carbohydrate supply to root and N transport to shoot), the assay must be conducted quickly to avoid such complications. Here we present a novel field method for simultaneous measurements of NH ₄ ⁺ and NO ₃ ^- uptake kinetics in intact root systems. The application of this method is demonstrated using two tree species; red maple (Acer rubrum) and sugar maple (Acer saccharum) and two crop species soybean (Glycine max) and sorghum (Sorghum bicolor). Plants were grown in open-top chambers at either ambient or elevated levels of atmospheric CO₂ at two separate US national sites involved in CO₂ research. Absolute values of net uptake rates and the kinetic parameters determined by our method were found to be in agreement with the literature reports. Roots of the crop species exhibited a greater uptake capacity for both N forms relative to tree species. Elevated CO₂ did not significantly affect kinetics of N uptake in species tested except in red maple where it increased root uptake capacity, V, for NH ₄ ⁺ . The application, reliability, advantages and disadvantages of the method are discussed in detail. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of inoculation ofPoa pratensis andTriticum aestivum with root-associated,N2-fixing Klebsiella,Enterobacter and Azospirillum

Strains ofKlebsiella pneumoniae, Klebsiella terrigena, Enterobacter agglomerans andAzospirillum lipoferum were compared as diazotrophic inoculants in association withPoa pratensis andTriticum aestivum. Each strain colonized both plants in numbers ranging from 10⁴ to 10⁷ bacteria per root, and electron microscopy and immunofluorescence staining of inoculated roots revealed bacteria mainly on root hairs. Indirect immunofluorescence with specific antifimbriae antibodies showed that the enteric bacteria expressed their fimbria in both associations. All associations were positive in an acetylene reduction test but only in half of them was atmospheric nitrogen transferred to the plant. In the inoculated plants, variable effects in the dry matter and N yields in both hosts were observed and no correlation was found between dry matter, nitrogen content or the amount of fixed nitrogen. In infected plants, the number of root hairs and lateral roots increased and the length of the zone of elongation decreased. The changes in root morphology were more evident in associations with enteric bacteria than with Azospirillum. The results give further evidence on the importance of bacterial adhesion in associative N₂ fixation and suggest that bacteria-induced physiological changes in plant roots may be more important than the amount of nitrogen transferred to the plant.     

Age-dependent changes in diastolic Ca and Na concentrations in dystrophic cardiomyopathy: Role of Ca entry and IP3

Duchenne muscular dystrophy (DMD) is a lethal X-inherited disease caused by dystrophin deficiency. Besides the relatively well characterized skeletal muscle degenerative processes, DMD is also associated with a dilated cardiomyopathy that leads to progressive heart failure at the end of the second decade. The aim of the present study was to characterize the diastolic Ca²⁺ concentration ([Ca²⁺]_d) and diastolic Na⁺ concentration ([Na⁺]_d) abnormalities in cardiomyocytes isolated from 3-, 6-, 9-, and 12-month old mdx mice using ion-selective microelectrodes. In addition, the contributions of gadolinium (Gd³⁺)-sensitive Ca²⁺ entry and inositol triphosphate (IP₃) signaling pathways in abnormal [Ca²⁺]_d and [Na⁺]_d were investigated. Our results showed an age-dependent increase in both [Ca²⁺]_d and [Na⁺]_d in dystrophic cardiomyocytes compared to those isolated from age-matched wt mice. Gd³⁺ treatment significantly reduced both [Ca²⁺]_d and [Na⁺]_d at all ages. In addition, blockade of the IP₃-pathway with either U-73122 or xestospongin C significantly reduced ion concentrations in dystrophic cardiomyocytes. Co-treatment with U-73122 and Gd³⁺ normalized both [Ca²⁺]_d and [Na⁺]_d at all ages in dystrophic cardiomyocytes. These data showed that loss of dystrophin in mdx cardiomyocytes produced an age-dependent intracellular Ca²⁺ and Na⁺ overload mediated at least in part by enhanced Ca²⁺ entry through Gd³⁺ sensitive transient receptor potential channels (TRPC), and by IP₃ receptors.