Proton/electron stoichiometry of mitochondrialbc 1 complex. Influence of pH and transmembrane δpH

The effect of pH and transmembrane pH on the efficiency of the proton pump of the mitochondrialbc ₁ complex bothin situ and in the reconstituted state was studied. In both cases the H⁺/e ^– ratio for vectorial proton translocation by thebc ₁ complex respiring at the steady state, under conditions in which the transmembrane pH difference (pH) represents the only component of the proton motive force (p), was significantly lower than that measured under level flow conditions. The latter amounts, at neutral pH, to 1 (2 including the scalar H⁺ release). In the reconstituted system steady-state pH was modulated by changing the intravesicular buffer as well as the intra/extra-liposomal pH. Under these conditions the H⁺/e^– ratio varied inversely with the pH. The data presented show that pH exerts a critical control on the proton pump of thebc ₁ complex. Increasing the external pH above neutrality caused a decrease of the level flowH ⁺/e ^– ratio. This effect is explained in terms of proton/electron linkage inb cytochromes.     

Functional reconstitution of photosystem I reaction center from cyanobacteriumSynechocystis sp PCC6803 into liposomes using a new reconstitution procedure

Photosystem I reaction center from the cyanobacteriumSynechocystis sp PCC6803 was reconstituted into phosphatidylcholine/phosphatidic acid liposomes. Liposomes prepared by reversephase evaporation were treated with various amounts of different detergents and protein incorporation was analyzed at each step of the solubilization process. After detergent removal the activities of the resulting proteoliposomes were measured. The most efficient reconstitution was obtained by insertion of the protein complex into preformed liposomes destabilized by saturating amounts of octylglucoside. In the presence of N-methylphenazonium methosulfate and ascorbic acid, liposomes containing the reaction center catalyzed a light-dependent net H⁺ uptake as measured by the 9-aminoacridine fluorescence quenching and the pH meter. An important benefit of the new reconstitution procedure is that it produces a homogeneous population of large-size proteoliposomes with a low ionic permeability and with a majority inwardly directed H⁺ transport activity. In optimal conditions, a light-induced pH of about 1.8 units could be sustained at 20C in the presence of valinomycin. In the absence of valinomycin, a back-pressure effect of an electrical transmembrane potential decreased both the rate and the extent of the H⁺ transport. The reaction center was also co-reconstituted with F₀F₁ H⁺-ATPases from chloroplasts and from the thermophilic bacterium, PS3. The coreconstituted system was shown to catalyze a light-dependent phosphorylation which could only be measured in the presence of a high concentration of PSI (low lipid/PSI ratios) while no pH could be detected.     

Electrogenic behavior of the human red cell Ca2+ pump revealed by disulfonic stilbenes

Summary A systematic study was made of the action of 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid (SITS) and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) on active Ca²⁺ transport of human erythrocytes. Pumping activity was estimated in inside-out vesicles (IOV's) by means of Ca²⁺-selective electrodes or use of tracer⁴⁵Ca²⁺. The stilbenes exhibited an approximately equal inhibitory potency and their action could be overcome by carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP) at low but not at high stilbene concentrations. In the absence of DIDS. Ca²⁺ transport was not affected upon addition of valinomycin, but it was appreciably reduced when vesicles were preincubated with low DIDS concentrations. Such an effect was strictly dependent on the external K⁺ concentration and it was abolished when valinomycin was added together with FCCP. Similar results were obtained using IOV's prepared from intact cells which had been previously exposed to the stilbene. The findings clearly demonstrate the presence in human red cells of a partially electrogenic Ca²⁺ pump, exchanging one Ca²⁺ ion for one proton.     

Interactions of inorganic mercury with phospholipid micelles and model membranes. A31P-NMR study

The binding of inorganic mercury Hg(II) to phospholipid headgroups has been investigated by phosphorus-31 nuclear magnetic resonance of phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylcholine (PC) in water micellar and multilamellar phases. HgCl₂ triggers the aggregation of phospholipid micelles, leading to a lipid-mercury precipitate that is no longer detectable by high-resolution³¹P-NMR. The remaining signal area corresponds to micelles in the soluble fraction and is a non-linear function of the initial mercury-to-lipid molar ratio. Kinetics of micelle aggregation are exponential for the first 15 min and show a plateau tendency after 120 min. Apparent Hg(H) affinities for phospholipid headgroups are in the order: PE > PS > PC. The same binding specificity is observed when HgCl₂ is added to (1:1) mixtures of different micelles (PE + PC; PS + PC). However, mercury binding to mixed micelles prepared with two lipids (PE/PC or PS/PC) induces the aggregation of both lipids. Hg(II) also leads to a³¹P-NMR chemical shift anisotropy decrease of PC, PS and mixed (1:1) PE/PC multilamellar vesicles and markedly broadens PS spectra. This indicates that HgCl₂ binding forces phospholipid headgroups to reorient and that the concomitant network formation leads to a slowing down of PS membrane collective motions. Formation of a gel-like lamellar phase characterized by a broad NMR linewidth is also observed upon HgCl₂ binding to PE samples both in fluid (L) or hexagonal (H_II) phases. The PE hexagonal phase is no longer detected in the presence of HgCl₂. Mixed PE/PC dispersions remain in the fluid phase upon mercury addition, indicating that no phase separation occurs. Addition of excess NaCl leads to the appearance of the non-reactive species HgCl _inf4 ^sup2– and induces the reversal of all the above effects.Abbreviations A(t) time-dependence of peak area - A₄₀ peak area at t=40 min - 1/ rate of peak area decrease - isotropic chemical shift - isotropic chemical shift change - chemical shift anisotropy - DPPC dipalmitoylphosphatidylcholine - Hg(II) inorganic mercury - NMR nuclear magnetic resonance - pCl –log [Cl^–] - PC phosphatidylcholine - PE phosphatidylethanolamine - PL phospholipid - PS phosphatidylserine - R_i mercury-to-lipid molar ratio - MLV multilamellar vesicles - SUV small unilamellar vesicles     

The role of the carboxyl and amino groups of polyene macrolides in their interactions with sterols and their selective toxicity. A P-NMR study

The permeability induced by amphotericin B and vacidin A derivatives in large unilamellar lipidic vesicles containing various sterols has been studied using the proton-cation exchange method and ³¹P-NMR spectroscopy. Derivatives which have a free ionizable carboxyl group induce biphasic ‘all or none’ permeability typical of channel-forming ionophores, whatever the sterol present. In sterol-free membranes, they have no significant activity. Derivatives which lack a free ionizable carboxyl group exhibit this channel-like mode of action only in membranes containing ergosterol or sterols with an alkyl side like that of ergosterol. In membranes containing cholesterol or sterol whose side-chain is alike, a slow and progressive permeability is observed at high concentrations. This activity is observed in sterol-free membranes as well. Derivatives containing sugars with substituted amino groups always have lower ionophoric activity than those which are unsubstituted. The greatest decrease in activity was observed for N-acetyl derivatives. Substitution of the amino groups has no effect on the mode of action. A model of interaction of polyenes with sterols is presented accounting for the data obtained on vesicles and the observed selective toxicity of polyene derivatives in biological membranes.     

Unilamellar-Vesicle Systems as Model for Studying Membrane Permeabilization by Polyene Antibiotics

Abstract

Permeabilization of phospholipid/sterol unilamellar vesicles by polyene antibiotics (amphotericin B and lucensomycin) was studied by measuring proton leakage with a pH-stat method. The percentage of proton release was directly related to the antibiotic concentration. Using ergosterol-containing vesicles, a relevant proton efflux was induced by micromolar concentrations of amphotericin B, whereas lucensomycin caused membrane permeabilization at higher concentrations (0.1 mM). Cholesterol-containing vesicles were less sensible to the lytic action of polyenes. When amphotericin B was carried in cholesterol-containing liposomes, the selectivity towards ergosterol-containing vesicles was enhanced. An increase in drug selectivity was also observed by dissolving amphotericin B in fresh human plasma. At concentrations one order of magnitude lower than those necessary to induce a detectable proton efflux, lucensomycin seemed to protect the vesicles from the subsequent permeabilizing action of amphotericin B.     

Subcellular localization of H+-ATPase from pumpkin hypocotyls (Cucurbita maxima L.) by membrane fractionation

A new method of preparing sealed vesicles from membrane fractions of pumpkin hypocotyls in ethanolamine-containing buffers was used to investigate the subcellular localization of H⁺-ATPase measured as nigericin-stimulated ATPase. In a fluorescence-quench assay, the H⁺ pump was directly demonstrated. The H⁺ pump was substrate-specific for Mg·ATP and 0.1 mM diethylstilbestrol completely prevented the development of a pH. The presence of unsupecific phosphatase hampered the detection of nigericin-stimulated ATPase. Unspecific phosphatases could be demonstrated by comparing the broad substrate specificity of the hydrolytic activities of the fractions with the clear preference for Mg·ATP as the substrate for the proton pump. Inhibitor studies showed that neither orthovanadate nor molybdate are absolutely specific for ATPase or acid phosphatase, respectively. Diethylstilbestrol seemed to be a specific inhibitor of ATPase activity in fractions containing nigericin-stimulated ATPase, but it stimulated acid phosphatase which tended to obscure its effect on ATPase activity. Nigericin-stimulated ATPase had its optimum at pH 6.0 and the nigericin effect was K⁺-dependent. The combination of valinomycin and carbonylcyanide m-chlorophenylhydrazone had a similar effect to nigericin, but singly these ionophores were much less stimulatory. After prolonged centrifugation on linear sucrose gradients, nigericin-stimulated ATPase correlated in dense fractions with plasma membrane markers but a part of it remained at the interphase. This lessdense part of the nigericin-stimulated ATPase could be derived from tonoplast vesicles because -mannosidase, an enzyme of the vacuolar sap, remained in the upper part of the gradient. Nigericinstimulated ATPase did not correlate with the mitochondrial marker, cytochrome c oxidase, whereas azide inhibition of ATPase activity did.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DES dethyltilbestrol     

15N natural abundances and N use by tundra plants

Plant species collected from tundra ecosystems located along a north-south transect from central Alaska to the north coast of Alaska showed large and consistent differences in ¹⁵N natural abundances. Foliar ¹⁵N values varied by about 10% among species within each of two moist tussock tundra sites. Differences in ¹⁵N contents among species or plant groups were consistent across moist tussock tundra at several other sites and across five other tundra types at a single site. Ericaceous species had the lowest ¹⁵N values, ranging between about –8 to –6. Foliar ¹⁵N contents increased progressively in birch, willows and sedges to maximum ¹⁵N values of about +2 in sedges. Soil ¹⁵N contents in tundra ecosystems at our two most intensively studied sites increased with depth and ¹⁵N values were usually higher for soils than for plants. Isotopic fractionations during soil N transformations and possibly during plant N uptake could lead to observed differences in ¹⁵N contents among plant species and between plants and soils. Patterns of variation in ¹⁵N content among species indicate that tundra plants acquire nitrogen in extremely nutrient-poor environments by competitive partitioning of the overall N pool. Differences in plant N sources, rooting depth, mycorrhizal associations, forms of N taken up, and other factors controlling plant N uptake are possible causes of variations in ¹⁵N values of tundra plant species.     

Foliar δ13C within a temperate deciduous forest: spatial,temporal, and species sources of variation

Summary Foliar ¹³C-abundance (¹³C) was analyzed in the dominant trees of a temperate deciduous forest in east Tennessee (Walker Branch Watershed) to investigate the variation in foliar ¹³C as a function of time (within-year and between years), space (canopy height, watershed topography and habitat) and species (deciduous and coniferous taxa). Various hypotheses were tested by analyzing (i) samples collected from the field during the growing season and (ii) foliar tissues maintained in an archived collection. The ¹³C-value for leaves from the tops of trees was 2 to 3%. more positive than for leaves sampled at lower heights in the canopy. Quercus prinus leaves sampled just prior to autumn leaf fall had significantly more negative ¹³C-values than those sampled during midsummer. On the more xeric ridges, needles of Pinus spp. had more positive ¹³C-values than leaves from deciduous species. Foliar ¹³C-values differed significantly as a function of topography. Deciduous leaves from xeric sites (ridges and slopes) had more positive ¹³C-values than those from mesic (riparian and cove) environments. On the more xeric sites, foliar ¹³C was significantly more positive in 1988 (a dry year) relative to that in 1989 (a year with above-normal precipitation). In contrast, leaf ¹³C in trees from mesic valley bottoms did not differ significantly among years with disparate precipitation. Patterns in foliar ¹³C indicated a higher ratio of net CO₂ assimilation to transpiration (A/E) for trees in more xeric versus mesic habitats, and for trees in xeric habitats during years of drought versus years of normal precipitation. However, A/E (units of mmol CO₂ fixed/mol H₂O transpired) calculated on the basis of ¹³C-values for leaves from the more xeric sites was higher in a wet year (6.6±1.2) versus a dry year (3.4±0.4). This difference was attributed to higher transpiration (and therefore lower A/E) in the year with lower relative humidity and higher average daily temperature. The calculated A/E values for the forest in 1988–89, based on ¹³C, were within ±55% of estimates made over a 17 day period at this site in 1984 using micrometeorological methods.     

Shoot δ15N correlates with genotype and salt stress in barley

Given a uniform N source, the ¹⁵N of barley shoots provided a genotypic range within treatments and a separation between control and salt-stress treatments as great as did ¹³C^*. Plant ¹⁵N has been represented in the literature as a bioassay of external source ¹⁵N and used to infer soil N sources, thus precluding consideration of the plant as a major cause in determining its own 8¹⁵N. We believe this to be the first report of plant ¹⁵N as a genetic trait. No mechanistic model is needed for use of ¹⁵N as a trait in controlled studies; however, a qualitative model is suggested for further testing.Symbol ¹⁵N (or ¹³C) the difference between: (1) the ratio of heavy to light isotopes of the element in a sample and (2) that of its reference standard     

Amiloride sensitivity of proton-conductive pathways in gastric and intestinal apical membrane vesicles

Summary Passive proton permeability of gastrointestinal apical membrane vesicles was determined. The nature of the pathways for proton permeation was investigated using amiloride. The rate of proton permeation (k _H ⁺ was determined by addition of vesicles (pH _i = 6.5) to a pH 8.0 solution containing acridine orange. The rate of recovery of acridine orange fluorescence after quenching by the acidic vesicles ranged from 4 × 10^–3 (gastric parietal cell stimulation-associated vesicles; SAV) and 5 × 10^–3 (duodenal brush-border membrane vesicles; dBBMV) to 11 × 10+^–3 sec^–1 (ileal BBMV; iBBMV). Amiloride, 0.03 and 0.1 mm, significantly reduced the rate of proton permeation in dBBMV and iBBMV, but not gastric SAV. The decreases in k _H ⁺ were proportionately greater in iBBMV as compared with dBBMV. The presence of Na⁺/H⁺ exchange was demonstrated in both dBBMV and iBBMV by proton-driven (pH _i < pH _o ) ²²Na⁺ uptake. Evidence was also sought for the conductive nature of pathways for proton permeation. Intravesicular acidification, again determined by quenching of acridine orange fluorescence, was observed during imposition of K⁺-diffusion potential ([K⁺] _i [K⁺ _o ). In dBBMV and iBBMV, intravesicular acidification was enhanced in the presence of the K⁺-ionophore valinomycin, indicating that the native K⁺ permeability is rate limiting. In the presence of valinomycin, the K⁺-diffusion potential drove BBMV intravesicular acidification to levels close to the electrochemical potential. In gastric SAV, acidification was not limited by the K⁺ permeability. Valinomycin was without effect, but the K⁺/H⁺ ionophore nigericin enhanced acidification in gastric SAV, illustrating the low proton permeability of these membranes. Amiloride, 0.03–1 mm, resulted in concentration-dependent reductions of K⁺-diffusion potential-driven acidification in dBBMV and iBBMV but not in gastric SAV. These data demonstrate that proton permeation in the three membrane types is rheogenic. The sensitivity of the proton-conductive pathways in intestinal BBMV to high concentrations of amiloride correlated with the presence of the Na⁺/H⁺ antiport and indicates that this transmembrane protein may represent a pathway for proton permeation.We thank Ruth Briggs for assistance with the Na/H exchange experiments. This work was supported by a grant from the Medical Research Council (G8418056CA).     

Ca2+ Sensitivity of Synaptic Vesicle Dopamine, γ-Aminobutyric Acid,and Glutamate Transport Systems

The effect of Ca2⁺ on the uptake of neurotransmitters by synaptic vesicles was investigated in a synaptic vesicle enriched fraction isolated from sheep brain cortex. We observed that dopamine uptake, which is driven at expenses of the proton concentration gradient generated across the membrane by the H⁺-ATPase activity, is strongly inhibited (70%) by 500 M Ca²⁺. Conversely, glutamate uptake, which essentially requires the electrical potential in the presence of low Cl^– concentrations, is not affected by Ca²⁺, even when the proton concentration gradient greatly contributes for the proton electrochemical gradient. These observations were checked by adding Ca²⁺ to dopamine or glutamate loaded vesicles, which promoted dopamine release, whereas glutamate remained inside the vesicles. Furthermore, similar effects were obtained by adding 150 M Zn²⁺ that, like Ca²⁺, dissipates the proton concentration gradient by exchanging with H⁺. With respect to -aminobutyric acid transport, which utilizes either the proton concentration gradient or the electrical potential as energy sources, we observed that Ca²⁺ or Zn²⁺ do not induce great alterations in the -aminobutyric acid accumulation by synaptic vesicles. These results clarify the nature of the energy source for accumulation of main neurotransmitters and suggest that stressing concentrations of Ca²⁺ or Zn²⁺ inhibit the proton concentration gradient-dependent neurotransmitter accumulation by inducing H⁺ pump uncoupling rather than by interacting with the neurotransmitter transporter molecules.     

Changes of the forest-savanna boundary in Brazilian Amazonia during the Holocene revealed by stable isotope ratios of soil organic carbon

The possibility of ecosystem boundary changes in northern Brazilian Amazonia during the Holocene period was investigated using soil organic carbon isotope ratios. Determination of past and present fluctuations of the forest-savanna boundary involved the measurement of natural ¹³C isotope abundance, expressed as ¹³C, in soil organic matter (SOM). SOM ¹³C analyses and radiocarbon dating of charcoal fragments were carried out on samples derived from soil profiles taken along transects perpendicular to the ecotonal boundary. SOM ¹³C values in the upper soil horizons appeared to be in equilibrium with the overlying vegetation types and did not point to a movement of the boundary during the last decades. However, ¹³C values obtained from deeper savanna and forest soil layers indicated that the vegetation type has changed in the past. In current savanna soil profiles, we observed the presence of mid-Holocene charcoals derived from forest species: fire frequency at that time was probably greater, and more extensive savanna may have resulted. Isotope data and the presence of these charcoals thus suggest that the forest-savanna boundary has shifted significantly in the recent Holocene period, forest being more extensive during the early Holocene than today. During the middle Holocene, the forest could have strongly regressed, and fires appeared, with a maximum development of the savanna vegetation. At the beginning of the late Holocene, the forest may have invaded a part of this savanna, and fires occurred again.     

Estimation of symbiotic dinitrogen fixation in alder forest by the method based on natural15N abundance

Annual N₂-fixation in virgin forest ecosystems has been measured using a¹⁵N natural abundance (¹⁵N) procedure. This method was compared to a¹⁵N labelled fertilizer isotopic dilution method. For young alders (5–6 years old), ¹⁵N of leaves gave results in good agreement with the isotopic dilution of fertilizer method. Since ¹⁵N variability was expected according to plant physiology, for alder trees, leaves were collected at various heights after the end of the growing season, and, to take account of isotopic variations coming from derived inputs, ¹⁵N of leaves of a large number of other plants in the same are were measured to give control values. Following this procedure, the ¹⁵N method gave reliable evaluation of the nitrogen supply, by through N₂-fixation, to alders, which were found to maintain high nitrogen fixing capacity in a sequence ranging from first stage of establishment of climactic formation. Moreover, the same method is reported to discriminate various origins ofAlnus glutinosa grown in natural conditions, possibly in relation to the genetic diversity of this species.     

Carbon isotope variations in a plantation of Sitka spruce,and the effect of acid mist

Intra- and inter-tree variations in ¹³C/¹²C ratios were studied within a single clone plantation of 20-year-old Sitka spruce, some of which were treated with mist simulating acidic cloud water. For groups of trees of similar height and the same treatment, sampled at the same whorl height, ¹³C values for current year needles showed variations (1 SD) of between 0.2 and 0.7. The variations reflect the seasonally averaged influences, on intercellular CO₂ concentrations, of slight variations in the microhabitat within a group. For a typical intra-group variation of 0.4 one may be able to distinguish between groups whose mean intercellular CO₂ concentrations differ by only 8 ppm. Acid misting resulted in a lowering of ¹³C values by c. 0.7 (significant at the P0.05 level). This reflects higher intercellular CO₂ concentrations for acid misted trees, which can be interpreted in terms of their having assimilation rates c. 10% lower than those of control trees, and might explain the observed reduction in stem growth for acid-misted trees. Without careful attention to sampling strategy, however, these small inter-tree ¹³C variations can be easily masked by the much larger intra-tree variations with height. Large gradients of increasing needle ¹³C with height, of c. 0.5 m^-1, were observed in two untreated trees of different total height. The gradient was similar for both trees so, though ¹³C values of both trees were identical close to their leaders (–27), the taller tree displayed much lower values close to the ground (–31). The gradients are believed to reflect lower light levels close to the ground, rather than the accumulation of respired CO₂ in the atmosphere. The different height response of stems versus needles, reflected by an increase in ¹³C_stems–¹³C_needles with height (for cellulose), is discussed in terms of stem photosynthetic recapture of internally respired CO₂.     

Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase exhibit proton translocating activity in the presence of gramicidin.

Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase (COV) were characterized for electron transfer and proton translocating activities in the presence of the mobile potassium ionophore, valinomycin, and the channel-forming ionophore, gramicidin, in order to determine if the ionophores modify the functional properties of the enzyme. In agreement with previous work, incubation of COV with valinomycin resulted in a perturbation of the absorbance spectrum of oxidized heme aa3 in the Soret region (430 nm); gramicidin had no effect on the heme aa3 absorbance spectrum. Different concentrations of the two ionophores were required for maximum respiratory control ratios in COV; 40- to 70-fold higher concentrations of valinomycin were required to completely uncouple electron transfer activity when compared to gramidicin. The proton translocating activity of COV incubated with each inophore gave a similar apparent proton translocated to electron transferred stoichiometry (H+/e- ratio) of 0.66 +/- 0.10. However, COV treated with low concentrations of gramicidin (0.14 mg/g phospholipid) exhibited 1.5- to 2.5-fold higher rates of alkalinization of the extravesicular media after the initial proton translocation reaction than did COV treated with valinomycin, suggesting that gramicidin allows more rapid equilibration of protons across the phospholipid bilayer during the proton translocation assay. Moreover, at higher concentrations of gramicidin (1.4 mg/g phospholipid), the observed H+/e- ratio decreased to 0.280 +/- 0.020, while the rate of alkalinization increased an additional 2-fold, suggesting that at higher concentrations, gramicidin acts as a proton ionophore. These results support the hypothesis that cytochrome c oxidase is a redox-linked proton pump that operates at similar efficiencies in the presence of either ionophore. Low concentrations of gramicidin dissipate the membrane potential in COV most likely by a channel mechanism that is different from the carrier mechanism of valinomycin, yet does not make the phospholipid bilayer freely permeable to protons.     

Influence of vegetation and soil CO2 exchange on the concentration and stable oxygen isotope ratio of atmospheric CO2 within a Pinus resinosa canopy

Measurements were made of the concentration and stable oxygen isotopic ratio of carbon dioxide in air samples collected on a diurnal basis at two heights within a Pinus resinosa canopy. Large changes in CO₂ concentration and isotopic composition were observed during diurnal time courses on all three symple dates. In addition, there was strong vertical stratification in the forest canopy, with higher CO₂ concentrations and more negative ¹⁸O values observed closer to the soil surface. The observed daily increases in ¹⁸O values of forest CO₂ were dependent on relative humidity consistent with the modelled predictions of isotopic fractionation during photosynthetic gas exchange. During photosynthetic gas exchange, a portion of the CO₂ that enters the leaf and equilibrates with leaf water is not fixed and diffuses back out of the leaf with an altered oxygen isotopic ratio. The oxygen isotope ratio of CO₂ diffusing out of a leaf depends primarily on the ¹⁸O content of leaf water which changes in response to relative humidity. In contrast, soil respiration caused a decline in the ¹⁸O values of forest CO₂ at night, because CO₂ released from the soil has equilibrated with soil water which has a lower ¹⁸O content than leaf water. The observed relationship between diurnal changes in CO₂ concentration and oxygen isotopic composition in the forest environment were consistent with a gas mixing model that considered the relative magnitudes of CO₂ fluxes associated with photosynthesis, respiration and turbulent exchange between the forest and the bulk atmosphere.     

Climatic adaptability of populations ofDiplotaxis erucoides D.C. (Brassicaceae) from Sicily,based on leaf morphology,leaf anatomy and δ13 C studies

The morphological and anatomical variability ofDiplotaxis erucoides populations from Sicily was investigated. Populations growing during the summer months exhibit distinct xeromorphic features. Leaf area is strongly reduced and leaf thickness is increased when compared with winter populations. Cell size, as well as cell arrangement and mesophyll cell surface area differ significantly between summer and winter populations. Leaf thickness is almost three times higher in summer populations andA (cell)/A, i.e. the mesophyll cell surface area per unit leaf area changes from about 16 for winter populations to almost 52 for summer populations. These differences are partly due to differences in intercellular volume and partly due to alterations in mesophyll cell sizes. The organic materal of the summer populations exhibits ¹³C values in the order of –27%. to –28%., while the corresponding values for the winter populations are in the order of –31%. to –33%.. Analysis ofD. erucoides populations from the transition period revealed intermediate ¹³C values. Anatomical variations such as reductions or increases ofA (cells)/A and changes of intercellular volume correlate with the corresponding ¹³C data. The ¹³C data are discussed in conjunction with the differences in leaf anatomy.     

Natural<Superscript>15</Superscript> N Abundance of Plants and Soil N in a Temperate Coniferous Forest

Measurement of nitrogen isotopic composition (¹⁵N) of plants and soil nitrogen might allow the characteristics of N transformation in an ecosystem to be detected. We tested the measurement of ¹⁵N for its ability to provide a picture of N dynamics at the ecosystem level by doing a simple comparison of ¹⁵N between soil N pools and plants, and by using an existing model. ¹⁵N of plants and soil N was measured together with foliar nitrate reductase activity (NRA) and the foliar NO₃^– pool at two sites with different nitrification rates in a temperature forest in Japan. ¹⁵N of plants was similar to that of soil NO₃^– in the high-nitrification site. Because of high foliar NRA and the large foliar NO₃^– pool at this site, we concluded that plant ¹⁵N indicated a great reliance of plants on soil NO₃^– there. However, many ¹⁵N of soil N overlapped each other at the other site, and ¹⁵N could not provide definitive evidence of the N source. The existing model was verified by measured ¹⁵N of soil inorganic N and it explained the variations of plant ¹⁵N between the two sites in the context of relative importance of nitrification, but more information about isotopic fractionations during plant N uptake is required for quantitative discussions about the plant N source. The model applied here can provide a basis to compare ¹⁵N signatures from different ecosystems and to understand N dynamics.     

Determination of the transmembrane proton gradient in the anaerobic bacterium Desulfovibrio desulfuricans by 31P nuclear magnetic resonance

The transmembrane proton gradient of the sulfate-reducing bacterium Desulfovibrio desulfuricans strain CSN has been determined by in vivo³¹P nuclear magnetic resonance (NMR) spectroscopy in the absence of dioxygen. At pH 7.0 in the medium (pH_ex) the intracellular pH (pH_in) was 7.5. By lowering pH_ex to 5.9 pH_in decreased to 7.1. At pH_ex greater than 7.7 the transmembrane proton gradient (pH) was zero. The uncouplers 3,3,4,5-tetrachlorosalicylanilide (TCS) and carbonylcyanide-m-chlorophenylhydrazone (CCCP), or the permeant anion thiocyanate caused complete dissipation of pH.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - TCS 3,3,4,5-tetrachlorosalicylanilide - MOPS 3-(N-morpholino)-propanesulfonic acid - P _i inorganic phosphate - pH _in (pH_ex) intracellular (extracellular) pH - pH transmembrane proton gradient (pH_in-pH_ex) - electrochemical membrane potential - chemical shift in parts per million - NMR nuclear magnetic resonance