15N estimates of nitrogen fixation by white clover (Trifolium repens L.) growing in a mixture with ryegrass (Lolium perenne L.)

The ¹⁵N isotope dilution technique and the N difference method were used to estimate N₂ fixation by clover growing in a mixture with ryegrass, in a field experiment and a controlled environment experiment. Values obtained using N difference were approximately 25% lower than those estimated using ¹⁵N isotope dilution. In the field experiment there was a measured N benefit to grass growing with clover, equivalent to 42.7 kgN ha^-1. The grass in the mixture had a lower atom %¹⁵N content and a higher N content than grass in a monoculture; therefore values for N₂ fixation were different depending on choice of control plant i.e. monoculture or mixture grass. In the controlled environment experiment there were no significant differences between either the atom %¹⁵N contents or the N contents of monoculture grass and grass growing in a mixture with clover. It is concluded that there is a long term indirect transfer of N from clover to associated grass which can lead to errors in estimates of N₂ fixation.     

Calcium uptake from the stigma by germinating pollen in Primula officinalis L. and Ruscus aculeatus L.

Summary The flow of calcium ions from the stigma to germinating pollen was studied by autoradiography in Primula officinalis (dry stigma) and Ruscus aculeatus (wet stigma). ⁴⁵Ca²⁺ ions were observed to be taken up by the pistils from an agar medium and then transported intracellularly to both the stigmal cells and the stigmal exudate. The ⁴⁵Ca²⁺ present in the stigma was taken up by the germinating pollen grains.     

A surgical method for accessing the plasmamembrane ofChara australis

Summary Progress in patch-clamping the plasmalemma ofChara australis has been slow because of the difficulty in obtaining a clean membrane surface upon which obtain a high resistance seal with the patch pipette. A surgical method is described here for cutting an access window in the cell wall of internodal cells fromChara australis. The method has a high success rate and also has the advantage of obviating the need for enzyme treatments. The patch-clamp technique is applied to theChara plasmalemma. Five types of channel activity are observed of which four of these are selective for Cl^– over K⁺.     

Molecular Cloning and Functional Analysis of a Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter Gene <Emphasis Type="Italic">ThNHX1</Emphasis> from a Halophytic Plant <Emphasis Type="Italic">Thellungiella halophila</Emphasis>

Na⁺/H⁺ exchanger catalyzes the countertransport of Na⁺ and H⁺ across membranes. Using the rapid amplification of cDNA ends method, a Na⁺/H⁺ antiporter gene (ThNHX1) was isolated from a halophytic plant, salt cress (Thellungiella halophila). The deduced amino acid sequence contained 545 amino acid residues with a conserved amiloride-binding domain (⁸⁷LFFIYLLPPI⁹⁶) and shared more than 94% identity with that of AtNHX1 from Arabidopsis thaliana. The ThNHX1 mRNA level was upregulated by salt and other stresses (abscisic acid, polyethylene glycol, and high temperature). This gene partially complemented the Na⁺/Li⁺-sensitive phenotype of a yeast mutant that was deficient in the endosomal–vacuolar Na⁺/H⁺ antiporter ScNHX1. Overexpression of ThNHX1 in Arabidopsis increased salt tolerance of transgenic plants compared with the wild-type plants. In addition, the silencing of ThNHX1 gene in T. halophila caused the transgenic plants to be more salt and osmotic sensitive than wild-type plant. Together, these results suggest that ThNHX1 may function as a tonoplast Na⁺/H⁺ antiporter and play an important role in salt tolerance of T. halophila. Chunxia Wu, Xiuhua Gao, and Xiangqiang Kong contributed equally to this work.     

Voltage-gated ion conductances corresponding to regenerative positive and negative spikes in the dinoflagellateNoctiluca miliaris

Depolarization-activated and hyperpolarization-activated ion conductances in the membrane of a marine dinoflagellateNoctiluca miliaris were examined under voltage-clamp conditions.Noctiluca exhibited a transient inward current in response to a step depolarization from a holding potential level of –80 mV to a potential level more positive than –50 mV. The I–V relationship for the current exhibited typical N-shaped characteristics similar to those of most excitable membranes. The current was inactivated by a membrane depolarization. The reversal potential of the current shifted in hyperpolarizing direction when the external Na⁺ concentration was lowered. The transient inward current is assumed to be responsible for the Na⁺-dependent positive spike in non-clamped specimens ofNoctiluca.Noctiluca exhibited a transient outward current in response to a step hyperpolarization from a holding potential level of –20 mV to a potential level more negative than –30 mV. The I–V relationship for the current was a typical N-shape as if it was turned 180° around its origin. The outward current showed a two-step exponential time-decay. The outward current was inactivated by a membrane hyperpolarization. The reversal potential shifted in the depolarizing direction when the external Cl^– concentration was lowered. The transient outward current is responsible for the Cl^–-dependent negative spike in non-clamped specimens ofNoctiluca.Abbreviations ASW artificial seawater - TRP tentacle regulating potentials - TTX tetrodotoxin     

Freemartinism and FecX allele determination in replacement ewes of the Rasa Aragonesa sheep breed by duplex PCR

A new naturally occurring mutation in the fecundity gene BMP15 in the Rasa Aragonesa sheep breed (Ovis aries) has been found to affect prolificacy. This mutation (FecX^R allele) is a deletion of 17 base pairs that leads to an altered amino acid sequence, and this alteration increases prolificacy in heterozygous ewes but causes sterility in homozygous ewes. Selection of repository lambs with the FecX^R allele increases rates of twins and multiple lambing and thereby also increases the probability of lambing freemartins that will become sterile. In this sense, an accurate, reliable, and quick method was developed by duplex polymerase chain reaction (PCR) for sex, amplifying an ovine-specific Y chromosome repetitive fragment, and BMP15 genotype determination in replacement ewe lambs. The BMP15 fragment served as an internal control of the amplification and detected the FecX^R allele, avoiding a false negative and then a mistake in freemartin detection. This assay uncovered 6 freemartin females among 195 replacement ewes from 7 different commercial flocks and 1 experimental flock. Furthermore, 1554 rams from 64 commercial flocks were also analyzed to identify FecX^R rams. This analysis identified 103 rams hemizygous for the FecX^R allele and 1 heterozygous ram. Because this gene is located on the X chromosome, this heterozygous animal is a freemartin ram that is co-amplifying the DNA from XX and XY lymphocytes. These results confirm the usefulness of this multiplex PCR assay for detecting phenotypically sexed females, freemartins, and the BMP15 genotype to detect highly prolific ewes in commercial flocks and to assist breeders in selection of repository lambs.     

Measurements of genetic variability for symbiotic dinitrogen fixation in fieldgrwon fababean (Vicia faba L.) using a low level15N-tracer technique

Before starting a breeding program aimed at improving the nitrogen nutrition ofVicia faba, the authors tried an alternative technique to the acetylene reduction assay, to measure some genetic variability in the plant material. The quantity of dinitrogen fixed by several cultivars ofVicia faba was estimated using a low enrichment¹⁵N tracer method and high precision¹⁵N mass spectrometry. The fababeans were cultivated for two years in two different soils. The percentage of fixed dinitrogen in the seed varied between genotypes from 40 to 83% of the total nitrogen and was positively correlated with the total seed nitrogen (r=0.64 to 0.86). A highly significant positive correlation was also found between the total seed nitrogen and the quantity of fixed dinitrogen in the seed (r=0.95 to 0.99). The technique used to measure dinitrogen fixation proved to be useful and reliable enough to discriminate between various genotypes, grown over a period of two years in two different soils. However, several non-fixing control plants showed significant differences in their¹⁵N enrichment and the problem of choosing a good reference plant was raised and discussed.     

Steady-State Proton Translocation in Bovine Heart Mitochondrial bc 1 Complex Reconstituted into Liposomes

The effect of different anions on the steady-state proton translocation in bovine bc ₁ complex reconstituted in liposomes was studied. The H⁺/e ^– ratio for vectorial proton translocation is at the steady state definitely lower than that measured at level flow, (0.3 vs. 1.0). The presence of azide or arachidonate at micro- and submicromolar concentrations, respectively, gave a substantial reactivation of the proton pumping activity at the steady state, without any appreciable effect on respiration-dependent transmembrane pH difference. Addition of azide to turning-over bc ₁ vesicles also caused a transition of b cytochromes toward oxidation. The results are discussed in terms of possible involvement of an acidic residue in the protonation of the semiquinone/quinol couple at the N side of the membrane.     

Influence of urea on biological N2 fixation and N transfer from Azolla intercropped with rice

The N₂ fixed by Azolla before and after urea application during the rice cycle, the mineralisation of Azolla-N as well as its availability to rice was studied in two greenhouse experiments conducted in 1996 and 1997 and in June 1998 in Goettingen (Germany). Dry matter production of the various rice parts of experiment 1 showed a clear positive synergism between treatment with Azolla and urea with a resulting apparent N recovery by rice increasing from 40% (without Azolla) to 57% in the presence of Azolla. Part of this increase may be due to N fixed biologically by Azolla and transferred to the rice. The second experiment shed some light on the role of BNF. Using an iterative method of estimation, the daily rate of N fixation was estimated at 0.6 – 0.7 kg N ha^–1. The rate was not so much affected by the age of the Azolla crop. At this rate, the BNF would amount to up to 100 kg N ha^–1 over a 130-day season. Assuming that BNF may be inhibited for a period of 5 – 10 days following urea application due to high levels of N in the floodwater, this might reduce the BNF by between 6 and 14 kg N ha over the season. Using the mean-pool-abundance concept, it was estimated that around 75 – 80% of the Azolla-N mineralized during the growth period was actually absorbed by the rice plants. Of the N taken up by rice around 28% was derived from the biologically fixed Azolla N, the remainder was urea N cycled through the Azolla. Azolla also seems to help sustain the soil N supply by returning N to the soil in quantities roughly equal to those extracted from the soil by the rice plant.     

A comparison between the acetylene reduction method,the isotope dilution method and the total nitrogen difference method for measuring nitrogen fixation in lucerne (Medicago sativa L.)

Summary In view of the increasing need for the exact estimation of the input of nitrogen in agroecosystems, an application of the acetylene-reduction technique was developed. The technique, consisting of a plastic bag incubation system, using propane as an internal standard of the apparent volume, made it possible to carry out repeated incubations on the same plant system. The evaluated technique included studies of the diffusion of ethylene and propane in a soil column, as well as studies of the optimal substrate concentration and grade of purity of the substrate. In addition, the conversion factor between amounts of reduced acetylene as compared wtih reduced dinitrogen was determined by¹⁵N₂ incubations to be 4.41. The developedin situ acetylene-reduction technique was compared with an isotope dilution method and a total nitrogen difference method. By comparing the derived total nitrogen fixation values from each with the value derived from the acetylene-reduction method; it was shown that the values differed significantly. The acetylene-reduction method gave the highest nitrogen fixation values, the isotope dilution the lowest values and the total nitrogen difference method was intermediate. No statistical significant difference existed between the two different reference crops used in the isotope dilution method.     

Stable Isotope Ratios as a Tool for Assessing Changes in Carbon and Nutrient Sources in Antarctic Terrestrial Ecosystems

Samples of an angiosperm species, nine lichen species and a terrestrial alga, were collected from a variety of Antarctic terrestrial habitats, and were analysed for C and N stable isotope composition. Collections were made along natural gradients, the marine gradient, running from the sea coast inland and the moisture gradient, determined by melt water and precipitation runoff, and running towards the sea coast. Considerable variation in stable isotope ratios was found; δ¹³C values ranged between −16 and −32‰ and δ¹⁵N values between −23 and +23‰ The variation in stable carbon isotope ratios could be attributed in part to species specific differences, but differences in water availability also played a role, as was shown for the terrestrial alga Prasiola crispa and the lichen species Usnea antarctica. The differences in the isotope ratios of nitrogen could be retraced to the origin of nitrogen: marine or terrestrial. The nitrogen stable isotope ratios were influenced by both the marine gradient from the sea inland and the melt water and precipitation flow running in the opposite direction, towards the sea. This was shown for the lichen species Turgidosculum complicatulum and the angiosperm species Deschampsia antarctica. The variation in the C and N stable isotope ratios can be used to determine sources and pathways of N and changes in the water availability in Antarctic terrestrial ecosystems. Contrary to earlier reports the use of stable N isotope ratios is possible in this case because of the relative simplicity of the structure of the Antarctic terrestrial ecosystems.     

克隆植物结缕草的水分生理整合格局特征及其生态效应分析

以克隆植物结缕草为研究对象,采用18 O作为示踪元素,从克隆植株不同生长发育阶段的复合节根系引入H218 O,在"异质高水"、"均质低水"两种环境条件下,探测和分析结缕草克隆植株复合节根、匍匐茎、A和B分株叶各构件组分系列内的水分生理整合格局特征及其生态效应。结果表明:(1)在两种水分环境条件下,H218 O在克隆植株主匍匐茎内各构件组分系列中均表现出双向传输的趋势,但更倾向于向顶传输。(2)H218 O向顶传输时,在"异质高水"生境内,基部复合节根系吸收的H218 O呈先增加后降低的趋势,而中部复合节根系吸收的H218 O呈先降低后增加的趋势;在"均质低水"生境内,中部复合节根系吸收的H218 O呈持续增加趋势。(3)在两种生境的3种引入情况下,H218 O均向顶传输到尖端生长点。其中在"异质高水"和"均质低水"生境内H218 O在克隆植株中向基传输过程中,传输强度整体上呈下降趋势;H218 O在主匍匐茎中传输时18 O分配于分株叶片中的量较多;H218 O在二级匍匐茎中的传输都呈现出明显的向顶趋势,传输距离都到达了二级匍匐茎的顶端生长点。(4)在绝大多数情况下,A分株叶系列的18 O丰度均明显高于B分株叶系列,这与A、B分株系列的生长发育特征相一致;但在"异质高水"生境内,中部分株吸收的H218 O在二级匍匐茎中传输时,分配于B分株叶系列的18 O明显高于A分株叶系列,即A分株系列相对于B分株系列的比较优势并不是一成不变的,在某些情况下还可以发生逆转。     

Evaluation of a short-incubation-time small-volume radiocarbon-uptake algal toxicity test

A short term small volume¹⁴C-assimilation algal toxicity test usingSelenastrum capricomutum CCAP 278/4 as test organism was used to determine the potential toxicity of a few selected compounds. Generally the sensitivity of the method increased with increasing exposure time to the substances tested. It was found that the method complied with the prerequisites of an acute test, because in most instances toxicity could be detected within 0.5 h exposure. Copper, cadmium, mercury and atrazine were the most toxic of the compounds tested.     

The use of a double isotope (15N and 34S) labelling technique to assess the suitability of various reference crops for estimating nitrogen fixation in Gliricidia sepium and Leucaena leucocephala

In this study, an approach involving a double isotope (¹⁵N and ³⁴S) labelling technique was used to examine which of five reference crops (Eucalyptus camaldulensis, Cassia siamea, Cassia spectabilis, Lolium perenne and Eucalyptus grandis) would be suitable for measuring N fixed by Gliricidia sepium and Leucaena leucocephala. The rationale is that the ratio of fertilizer-derived S to soil-derived S in a suitable reference crop is similar to that measured in the nitrogen fixing tree (NFT) since the N ratios in the two crop types cannot be measured directly. E. camaldulensis and E. grandis were found to be suitable reference crops because they absorbed fertilizer and soil S in the same ratio as G. sepium and L. leucocephala.     

Determination of 8-oxoguanine in human plasma and urine by high-performance liquid chromatography with electrochemical detection

A highly sensitive and selective method for determining 8-oxoguanine in plasma and urine was developed by high-performance liquid chromatography with electrochemical detection. The compound was separated by gradient elution on a C₁₈ reversed-phase column with a mobile phase of acetonitrile and 0.1 M sodium acetate, pH 5.2. 8-Hydroxy-2′-deoxyguanosine was used as internal standard. 8-Oxoguanine was detected electrochemically by setting the potential to +300 mV vs. Pd reference. The sensitivity of the assay was 22 ng/ml with a signal-to-noise ratio of 7:1. The within-day relative standard deviations for 8-oxoguanine quality control samples with concentrations of 3340, 1340 and 84 ng/ml were 3.6, 4.3 and 5.7% for plasma, and 4.1, 4.6 and 6.2% for urine, respectively. The day-to-day relative standard deviations for the same samples were 3.8, 6.8 and 7.1% for plasma, and 3.9, 7.0 and 7.9% for urine, respectively. The method is designed to study the pharmacokinetics and metabolic fate of O⁶-benzylguanine in a phase I clinical trial. Previously, O⁶-benzyl-8-oxoguanine was identified as the primary metabolite of O⁶-benzylguanine in humans. We now demonstrate that 8-oxoguanine is a further metabolite of O⁶-benzylguanine.     

Presence of a Na+/H+ exchanger in brush border membranes isolated from the kidney of the spiny dogfish,Squalus acanthias

Summary A membrane fraction, rich in brush border membranes, was prepared from renal proximal tubules of the spiny dogfish,Squalus acanthias, and the sodium-proton exchange mechanism in these membrane vesicles was investigated by both a rapid filtration technique and the fluorescence quenching of acridine organe.²²Na⁺ uptake was stimulated by an outwardly directed H⁺ gradient, and was inhibited by amiloride at a single inhibitory site with an apparentK _i of approximately 1.7×10^–5 M. In the presence of an H _i ⁺ >H _o ⁺ gradient, the of the Na⁺/H⁺ exchanger were 9.7±0.8 mM and 48.0±12.0 nmol·mg protein^–1·min^–1, respectively. The uptake of Na⁺ was electroneutral in the presence of a H⁺ gradient, indicating a stoichiometry of 1. In the fluorescence studies, quenching of acridine orange occurred in the presence of an outwardly directed Na⁺ gradient which was inhibited by amiloride. Thus, an electroneutral Na⁺/H⁺ exchanger with properties similar to those found in the mammalian kidney is also present in the spiny dogfish and may contribute to the urinary acidification of this marine animal.     

Cloning of two SOS1 transporters from the seagrass <Emphasis Type="Italic">Cymodocea nodosa</Emphasis>. SOS1 transporters from <Emphasis Type="Italic">Cymodocea</Emphasis> and <Emphasis Type="Italic">Arabidopsis</Emphasis> mediate potassium uptake in bacteria

Two cDNAs isolated from Cymodocea nodosa, CnSOS1A, and CnSOS1B encode proteins with high-sequence similarities to SOS1 plant transporters. CnSOS1A expressed in a yeast Na⁺-efflux mutant under the control of a constitutive expression promoter mimicked AtSOS1 from Arabidopsis; the wild type cDNA did not improve the growth of the recipient strain in the presence of Na⁺, but a cDNA mutant that expresses a truncated protein suppressed the defect of the yeast mutant. In similar experiments, CnSOS1B was not effective. Conditional expression, under the control of an arabinose responsive promoter, of the CnSOS1A and CnSOS1B cDNAs in an Escherichia coli mutant defective in Na⁺ efflux was toxic, and functional analyses were inconclusive. The same constructs transformed into an E. coli K⁺-uptake mutant revealed that CnSOS1A was also toxic, but that it slightly suppressed defective growth at low K⁺. Truncation in the C-terminal hydrophilic tail of CnSOS1A relieved the toxicity and proved that CnSOS1A was an excellent low-affinity K⁺ and Rb⁺ transporter. CnSOS1B mediated a transient, extremely rapid K⁺ or Rb⁺ influx. Similar tests with AtSOS1 revealed that it was not toxic and that the whole protein exhibited excellent K⁺ and Rb⁺ uptake characteristics in bacteria.     

Molecular characterization of PeSOS1: the putative Na+/H+ antiporter of Populus euphratica

Populus euphratica is a salt-tolerant tree species growing in semi-arid saline areas. A Na⁺/H⁺ antiporter gene was successfully isolated from this species through RACE cloning, and named PeSOS1. The isolated cDNA was 3665 bp long and contained a 3438 bp open reading frame that was predicted to encode a 127-kDa protein with 12 hypothetical transmembrane domains in the N-terminal part and a long hydrophilic cytoplasmic tail in the C-terminal part. The amino acid sequence of this PeSOS1 gene showed 64% identity with the previously isolated SOS1 gene from the glycophyte Arabidopsis thaliana. The level of protein expressed by PeSOS1 in the leaves of P. euphratica was significantly up-regulated in the presence of high (200 mM) concentrations of NaCl, while the mRNA level in the leaves remained relatively constant. Immunoanalysis suggested that the protein encoded by PeSOS1 is localized in the plasma membrane. Expression of PeSOS1 partially suppressed the salt sensitive phenotypes of the EP432 bacterial strain, which lacks the activity of the two Na⁺/H⁺ antiporters EcNhaA and EcNhaB. These results suggest that PeSOS1 may play an essential role in the salt tolerance of P. euphratica and may be useful for improving salt tolerance in other tree species. Yuxia Wu and Nan Ding contributed equally to this work.     

Development of spatio-temporal pattern of Ca2+ on the chemotactic behaviour ofPhysarum plasmodium

Summary The development of a spatio-temporal pattern of Ca²⁺ concentration by a plasmodium ofPhysarum polycephalum during chemotaxis was studied using fura-2. Whenever the cell displayed coordinated migration in one direction as a whole body, a spatiotemporal pattern was established with a characteristic feature along the longitudinal axis. Calcium concentration oscillated with a period of a few minutes within the cell; the mean concentration at the front was higher than that at the rear. When the cell was given an attractant only at the rear end, the mean concentration rose at the site of application with an immediate increase in the frequency of oscillation. First, the change of the frequency is propagated toward the other end and then the mean level of the Ca²⁺ concentration at the non-stimulated site decreases. As a result, the Ca²⁺ gradient is reversed along the cell, which then begins to migrate in a coordinated manner in the reverse direction. This study showed that the spatiotemporal pattern of Ca²⁺ concentration is closely related to information processing for coordinated migration in chemotaxis. The role of the pattern in that process is discussed.