The Use of the [13C]/[12C] Ratio for the Assay of the Microbial Oxidation of Hydrocarbons

The study deals with a comparative analysis of the relative abundances of the carbon isotopes ¹²C and ¹³C in the metabolites and biomass of the Burkholderia sp. BS3702 and Pseudomonas putida BS202-p strains capable of utilizing aliphatic (n-hexadecane) and aromatic (naphthalene) hydrocarbons as sources of carbon and energy. The isotope compositions of the carbon dioxide, biomass, and exometabolites produced during the growth of Burkholderia sp. BS3702 on n-hexadecane (¹³C = –44.6 ± 0.2) were characterized by the values of ¹³C_{CO 2} = –50.2 ± 0.4, ¹³C_biom = –46.6 ± 0.4, and ¹³C_exo = –41.5 ± 0.4, respectively. The isotope compositions of the carbon dioxide, biomass, and exometabolites produced during the growth of the same bacterial strain on naphthalene (¹³C = –21 ± 0.4) were characterized by the isotope effects ¹³C_{CO 2} = –24.1 ± 0.4, ¹³C_biom = –19.2 ± 0.4, and ¹³C_exo = –19.1 ± 0.4, respectively. The possibility of using the isotope composition of metabolic carbon dioxide for the rapid monitoring of the microbial degradation of petroleum hydrocarbons in the environment is discussed.     

Solution conformations of proline rings in proteins studied by NMR spectroscopy

Summary Three different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine -, - and -hydrogens have been made on the basis of ¹H-¹H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs -³, ³-³, ²-², ²-², and ³-³ (2.3 Å) are shorter than those in the pairs -², ²-³, ³-², ²-³, and ³-² (2.7–3.0 Å), resulting in stronger NOESY cross peaks. For the Up conformation, the ³-² and ²-³ spin-spin coupling constants are small (<3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the -², ²-³, and ³-² vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.     

Using stable isotopes to investigate migratory connectivity of the globally threatened aquatic warbler Acrocephalus paludicola

Understanding the links between breeding and wintering areas of migratory species has important ecological and conservation implications. Recently, stable isotope technology has been used to further our understanding. Stable isotope ratios vary geographically with a range of biogeochemical factors and isotope profiles in organisms reflect those in their food and environment. For inert tissues like feathers, isotope profiles reflect the environment in which they were formed. Following large-scale habitat destruction, the globally threatened aquatic warbler Acrocephalus paludicola has a fragmented breeding population across central Europe, largely in Belarus, Poland and Ukraine. The species sub-Saharan African wintering grounds have not yet been discovered, and this significantly hampers conservation efforts. Aquatic warblers grow their flight feathers on their wintering grounds, and we analysed stable isotope ratios (¹⁵N, ¹³C, D) in rectrices of adults from six main breeding sites (subpopulations) across Europe to determine whether different breeding subpopulations formed a single mixed population on the wintering grounds. ¹⁵N varies considerably with dietary trophic level and environmental factors, and D with the D in rainfall; neither varied between aquatic warbler subpopulations. Uniform feather ¹⁵N signatures suggest no major variation in dietary trophic level during feather formation. High variance and inter-annual differences in mean D values hinder interpretation of these data. Significant differences in mean ¹³C ratios existed between subpopulations. We discuss possible interpretations of this result, and consider differences in moulting latitude of different subpopulations to be the most parsimonious. ¹³C in plants and animals decreases with latitude, along a steep gradient in sub-Saharan Africa. Birds from the most north-westerly breeding subpopulation (Karsibor, Poland) had significantly lower variance in ¹³C and ¹⁵N than birds from all other sites, suggesting either that birds from Karsibor are less geographically dispersed during moult, or moult in an area with less isotopic heterogeneity. Mean ¹³C signatures from winter-grown feathers of different subpopulations were positively correlated with the latitude and longitude of breeding sites, suggesting a strong relationship between European breeding and African winter moulting latitudes. The use of stable isotopes provides novel insights into migratory connectivity and migration patterns in this little-known threatened species.     

15N natural abundance studies in Australian commercial sugarcane

The measurement of natural ¹⁵N abundance is a well-established technique for the identification and quantification of biological N₂ fixation in plants. Associative N₂ fixing bacteria have been isolated from sugarcane and reported to contribute potentially significant amounts of N to plant growth and development. It has not been established whether Australian commercial sugarcane receives significant input from biological N₂ fixation, even though high populations of N₂ fixing bacteria have been isolated from Australian commercial sugarcane fields and plants. In this study, ¹⁵N measurements were used as a primary measure to identify whether Australian commercial sugarcane was obtaining significant inputs of N via biological N₂ fixation. Quantification of N input, via biological N₂ fixation, was not possible since suitable non-N₂ fixing reference plants were not present in commercial cane fields. The survey of Australian commercially grown sugarcane crops showed the majority had positive leaf ¹⁵N values (73% >3.00, 63% of which were >5.00), which was not indicative of biological N₂ fixation being the major source of N for these crops. However, a small number of sites had low or negative leaf ¹⁵N values. These crops had received high N fertiliser applications in the weeks prior to sampling. Two possible pathways that could result in low ¹⁵N values for sugarcane leaves (other than N₂ fixation) are proposed; high external N concentrations and foliar uptake of volatilised NH₃. The leaf ¹⁵N value of sugarcane grown in aerated solution culture was shown to decrease by approximately 5 with increasing external N concentration (0.5–8.0 mM), with both NO₃ ^– and NH₄ ⁺ nitrogen forms. Foliar uptake of atmospheric NH₃ has been shown to result in depleted leaf ¹⁵N values in many plant species. Acid traps collected atmospheric N with negative ¹⁵N value (–24.45±0.90) from above a field recently surface fertilised with urea. The ¹⁵N of leaves of sugarcane plants either growing directly in the soil or isolated from soil in pots dropped by 3.00 in the same field after the fertiliser application. Both the high concentration of external N in the root zone (following the application of N-fertilisers) and/or subsequent foliar uptake of volatilised NH₃ could have caused the depleted leaf ¹⁵N values measured in the sugarcane crops at these sites.     

Natural abundance of 15N in tropical plants with emphasis on tree legumes

Natural abundance of ¹⁵N ( ¹⁵N) of leaves harvested from tropical plants in Brazil and Thailand was analyzed. The ¹⁵N values of non-N₂-fixing trees in Brazil were +4.5±1.9, which is lower than those of soil nitrogen (+8.0±2.2). In contrast, mimosa and kudzu had very low ¹⁵N values (–1.4+0.5). The ¹⁵N values of Panicum maximum and leguminous trees, except Leucaena leucocephala, were similar to those of non-N₂-fixing trees, suggesting that the contribution of fixed N in these plants is negligible. The ¹⁵N values of non-N₂-fixing trees in Thailand were +4.9±2.0. Leucaena leucocephala, Sesbania grandiflora, Casuarina spp. and Cycas spp. had low ¹⁵N values, close to the value of atmospheric N₂ (0), pointing to a major contribution of N₂ fixation in these plants. Cassia spp. and Tamarindus indica had high ¹⁵N values, which confirms that these species are non-nodulating legumes. The ¹⁵N values of Acacia spp. and Gliricidia sepium and other potentially nodulating tree legumes were, on average, slightly lower than those of non-N₂-fixing trees, indicating a small contribution of N₂ fixation in these legumes.     

Detection of very weak side chain–main chain hydrogen bonding interactions in medium-size 13C/15N-labeled proteins by sensitivity-enhanced NMR spectroscopy

We describe the direct observation of very weak side chain–main chain hydrogen bonding interactions in medium-size ¹³C/¹⁵N-labeled proteins with sensitivity-enhanced NMR spectroscopy. Specifically, the remote correlation between the hydrogen acceptor side chain carboxylate carbon ¹³CO₂ of glutamate 54 and the hydrogen donor backbone amide ¹⁵N of methionine 49 in a 12 kDa protein, human FKBP12, is detected via the trans-hydrogen bond ^3h J _NCO2 coupling by employing a novel sensitivity-enhanced HNCO-type experiment, CPD-HNCO. The ^3h J _NCO2 coupling constant appears to be even smaller than the average value of backbone ^3h J _NC couplings, consistent with more extensive local dynamics in protein side chains.     

Using stable hydrogen and oxygen isotope measurements of feathers to infer geographical origins of migrating European birds

Successful application of stable-hydrogen isotope measurements (D_f) of feathers to track origins of migratory birds and other wildlife requires a fundamental understanding of the correlation between D_f and deuterium patterns in rainfall (D_p) over continental scales. A strong correlation between D_p and D_f has been confirmed for birds and insects in North America, but not yet for other continents. Here, we compare D_f data from resident European birds to new D_p basemaps for Europe. Three maps, representing growing-season and mean annual D_p estimates from an elevation-explicit, detrended interpolation model and growing-season D_p estimates from simple Kriging, all indicate that strong isotope gradients occur across Europe with a general depletion occurring in a northeast direction. The feather data, representing 141 individuals of 25 avian species from 38 sites, ranged from –131 to –38. Regression analysis showed that strong correlations existed between both mean annual and growing-season D_p estimated by detrended interpolation and D_f of non-aquatic and non-corvid birds (r²=0.66 and 0.65, respectively). We also examined mean annual and growing-season ¹⁸O_p vs. ¹⁸O_f for our samples. Both oxygen regressions were similar (r²=0.56 and 0.57, respectively) but poorer than for deuterium. Our study reveals that D measurements of feathers from migratory birds in Europe may be used to track their origin and movements, and so provide a powerful investigative tool for avian migration research in Europe.     

Mapping of Exposed Surfaces of the Nicotinic Acetylcholine Receptor by Identification of Iodinated Tyrosine Residues

Here we report on the use of iodination of the membrane-bound nicotinic acetylcholine receptor (nAChR) from Torpedo californica electric tissue in order to define surface-exposed portions of the receptor molecule. Membrane-bound nAChR was ¹²⁵I-iodinated using the oxidation agent Iodo-Gen. The iodinated subunits were separated by preparative gel electrophoresis, desalted, and cleaved with trypsin. The resulting peptides were separated by reverse-phase HPLC and the radioactive peptides were identified by mass spectrometry and protein sequencing. For the -subunit, we identified five iodinated peptides containing the tyrosine residues Tyr17, Tyr74, Tyr365, Tyr372, and Tyr428. The surface exposition of these amino acids is in agreement with the four-transmembrane-segment model (4TM model) of the nAChR, but the assignment to the intra- or extracellular surface is doubtful. According to this model, the N-terminal portion of the receptor subunits including the iodinated residues Tyr17 and Tyr74 is extracellular and Tyr372 as a site of tyrosine phosphorylation is located on the cytoplasmic side. But since this latter residue is among the first to be iodinated using an immobilized iodination agent, its true position with respect to the membrane bilayer is not clear.     

Mean oxygen-isotope signatures in Porites spp. corals: inter-colony variability and correction for extension-rate effects

The assessment of inter-colony variability in the mean skeletal ¹⁸O signatures of modern Porites spp. corals is a prerequisite for the estimation of past mean climate conditions based on fossil colonies. Here we show that the mean ¹⁸O signatures of Porites spp. corals from the northern end of the Gulf of Aqaba (Red Sea) with mean extension rates between 0.2 and 1.5 cm/year can have an inter-colony variability as large as 1.28. At extension rates of less than 0.6 cm/year the mean coral ¹⁸O values of the individual colonies are strongly dependent on the mean extension rate, with increasingly higher ¹⁸O values corresponding to decreasing extension rate. This suggests that extension-rate-related kinetic isotope disequilibrium effects are responsible for a large proportion of the inter-colony differences in the mean coral ¹⁸O signatures. A correction procedure for these effects based on the relationship between mean ¹⁸O values and mean extension rate reduces the variability of mean coral ¹⁸O values among the individual colonies to 0.43. Although certainly not perfect, the correction procedure enables a better assessment of mid-Holocene climate conditions at this location based on Porites spp. with mean extension rates of less than 0.6 cm/year.     

Spatial and temporal distribution of carbon isotopes in soil organic matter at the Dinghushan Biosphere Reserve, South China

The spatial and temporal distribution of carbon isotopes (¹³C, ¹⁴C) in soil organic matter (SOM) were studied based on SOM content, SOM ¹⁴C and SOM ¹³C of thinly layered soil samples for six soil profiles with different elevations at the Dinghushan Biosphere Reserve (DHSBR), South China. The results indicate that variations of SOM ¹³C with depth of the soil profiles at different elevations are controlled by soil development, and correlate well with SOM composition in terms of SOM compartments with different turnover rates, and SOM turnover processes at the DHSBR. The effect of carbon isotope fractionation was obvious during transformation of organic matter (OM) from plant debris to SOM in topsoil and SOM turnover processes after the topsoil was buried, which resulted in great increments of OM ¹³C, respectively. Increments of SOM ¹³C of topsoil from ¹³C of plant debris were controlled by SOM turnover rates. Both topsoil SOM ¹³C and plant debris ¹³C increase with elevation, indicating regular changes in vegetation species and composition with elevation, which is consistent with the vertical distribution of vegetation at the DHSBR. The six soil profiles at different elevations had similar characteristics in variations of SOM ¹³C with depth, alterations of SOM contents with depth and that SOM ¹⁴C apparent ages increasing with depth, respectively. These are presumably attributed to the regular distribution of different SOM compartments with depth because of their regular turnover during soil development. Depth with the maximal SOM ¹³C value is different in mechanism and magnitude with penetrating depth of ¹⁴C produced by nuclear explosion into atmosphere from 1952 to 1962, and both indicate controls of topography and vegetation on the distribution of SOM carbon isotopes with depth. Elevation exerts indirect controls on the spatial and temporal distribution of SOM carbon isotopes of the studied mountainous soil profiles at the DHSBR. This study shows that mountainous soil profiles at different elevations and with distinctive aboveground vegetation are presumably ideal sites for studies on soil carbon dynamics in different climatic-vegetation zones.     

Heterogeneous binding and killing behaviour of human γ/δ-TCR+ lymphokine-activated killer cells against K562 and Daudi cells

Effector-target conjugates, formed by coincubation of lymphokine-activated killer (LAK) cells with either K562 or Daudi cells, were separated from single cells by Percoll sedimentation. The occurrence of various CD molecules (CD3, CD56, CD57, CD16, /-TCR) was compared in both fractions. Only LAK cells expressing the / T cell receptor (TCR) were found in a significantly increased percentage in fractions containing conjugates indicating that /-TCR⁺ LAK cells were preferably bound to target cells at the time of separation. In order to determine whether /-TCR⁺ LAK cells also show a preferred killing activity against the targets, cultures enriched with or depleted of /-TCR⁺ cells were established. Against K562 cells, /-TCR⁺-enriched cultures showed a greatly reduced killing activity compared to LAK bulk cultures or cultures depleted of /-TCR⁺ cells. Using Daudi cells as targets the enriched fraction revealed a slightly increased killing activity compared to bulk cultures or depleted fractions. Preincubation of /-TCR⁺ LAK cells with anti-/ or anti-CD3 mAb resulted in a distinct increase of the killing activity against K562 cells, but in only a slightly enhanced activity against Daudi cells. It is postulated that /-TCR⁺ LAK cells use the same adhesion mechanism for both targets but that only Daudi cells express a specific ligand for the /-TCR. Occupation of the /-TCR/CD3 complex by mAb, however, seems to substitute for the absent epitope on K562 cells by eliciting stimulatory signals in /-TCR⁺ LAK cells which, in combination with the binding stimulus, trigger cytolytic activity.This work was supported by the Hartmann-Müller Foundation, Zürich     

The 15N/14N ratios of plants in South Africa and Namibia: relationship to climate and coastal/saline environments

Summary Data are presented for the ¹⁵N/¹⁴N ratios of 140 indigenous terrestrial plants from a wide variety of natural habitats in South Africa and Namibia. Over much of the area, from high-rainfall mountains to arid deserts, the ¹⁵N values of plants lie typically in the range -1 to +6; with no evident differences between C₃ plants and C₄ grasses. There is a slight correlation between ¹⁵N and aridity, but this is less marked than the correlation between the ¹⁵N values of animal bones and aridity. At coastal or saline sites, however, the mean ¹⁵N values for plants are higher than those at nearby inland or non-saline sites-e.g.: arid Namib coast (10 higher than inland Namib); wet Natal beach (5 higher than inland Natal); saline soils 500 km from coast (4 higher than non-saline soils). High values were also found at one site where there were no marked coastal or saline influences. These environmental effects on the isotopic composition of plants will extend upwards to the animals and humans they support. They therefore have important consequences for the use of nitrogen isotope data in the study of the dietary habits and trophic structures of modern and prehistoric communities.     

Stimulation of μ- and δ-Opiate Receptors and Tolerance of Isolated Heart to Oxidative Stress: the Role of NO-Synthase

Preliminary intravenous injection of -opiate receptor (OR) agonists DSLET (0.1 mg/kg) or DTLET (0.1 mg/kg) increased tolerance of isolated perfused myocardium to damage by oxidative stress simulated in vivo with FeSO₄ + ascorbic acid. This manifested itself by a decreased level of creatine phosphokinase (CPK) in the perfusate flowing out of the heart during the oxidative exposure. The preliminary systemic injection of -agonists DAMGO (0.1 mg/kg) or DALDA (0.1 mg/kg) failed to affect the release of CPK from the myocardium. The cardioprotective effect of the -agonist DSLET was completely abolished by preliminary intravenous injection of the -OR antagonist ICI 174,864 (2.5 mg/kg). The intravenous injection of the NO-synthase inhibitor L-NAME (50 mg/kg) also completely abolished the cardioprotective effect of -OR stimulation. The preliminary injection of DSLET but not of DAMGO prevented an increase in the level of diene conjugates and a decrease in the activity of superoxide dismutase (SOD) in the isolated myocardium tissue. Thus, the in vivo stimulation of -OR increased the tolerance of the heart to oxidative stress through activation of NO-synthase and SOD.     

Autotrophic carbon sources for heterotrophic bacterioplankton in a floodplain lake of central Amazon

The relative contribution of autotrophic carbon sources (aquatic macrophytes, flooded forest, phytoplankton) for heterotrophic bacterioplankton was evaluated in a floodplain lake of the Central Amazon. Stable carbon isotopes (¹³C) were used as tracers. Values of ¹³C of different autotrophic sources were compared to those of dissolved organic carbon (DOC) and those of bacterially produced CO₂.The percentage of carbon derived from C₄ macrophytes for bacterially produced CO₂ was the highest, on average 89%. The average ¹³C value of CO₂ from bacterial respiration was –18.5 ± 3.3. Considering a fractionation of CO₂ of 3 by bacterial respiration, ¹³C value was –15.5, near C₄ macrophyte ¹³C value (–13.1).The average value of total DOC ¹³C was –26.8 ± 2.4. The percentage of C₄ macrophytes carbon for total DOC was on average 17%. Considering that bacteria consume mainly carbon from macrophytes, the dominance of C₃ plants for total DOC probably reflects a faster consumption of the former source, rather than a major contribution of the latter source.Heterotrophic bacterioplankton in the floodplain may be an important link in the aquatic food web, transferring the carbon from C₄ macrophytes to the consumers.     

Preliminary studies of the impact of excreted N on cycling and uptake of N in pasture systems using natural abundance stable isotopic discrimination

The possibility of using natural abundance techniques to determine N transformations and flows after deposition of cattle dung has been examined. These preliminary results showed that ¹⁵N in dung was greater than in plants growing in association with particular pats. This, and other observational information, indicated that dung pats of different ages were being examined. There were significant variations in plant ¹⁵N signatures within and between species grown in association and away from the dung. It was probable that variation in plant ¹⁵N was brought about by changes in soil mineral N pools after transfer of N derived from the dung. This resulted in different ¹⁵N signatures in Trifolium repens (because of changes in N utilization from soil or atmospheric pools), in Lolium perenne (because of changes in ¹⁵N in soil mineral N), but not in Ranunculus repens (because the majority of active roots were outside the range of immediate influence of the deposited dung). The differences in ¹⁵N allowed the development of hypotheses for changes in soil N pools and the acquisition of N by plants from soil, dung or atmospheric sources.     

A simple urea leaf-feeding method for the production of 13C and 15N labelled plant material

The use of ¹³C isotope tracer techniques in terrestrial ecology has been restricted by the technical requirements and high costs associated with the production of ¹³C enriched plant material by ¹³CO₂ release in labelling chambers. We describe a novel, simple and relatively inexpensive method for the small-scale production of ¹³C and ¹⁵N labelled plant material. The method is based on foliar feeding of plants with a urea solution (97 atom% ¹³C, 2 atom% ¹⁵N) by daily misting. Maize was grown in a greenhouse in a compost–soil mixture and enclosed in clear polythene bags between urea applications. Final enrichment in 27 d old maize shoots was 211 ¹³C (1.34 atom% ¹³C) and 434 ¹⁵N (0.52 atom% ¹⁵N). Enrichments of hot-water extractable fractions (289 ¹³C, 469 ¹⁵N) were only slightly higher than those observed in plant bulk material, which suggests that daily urea applications ensured fairly uniform labelling of different biochemical fractions and plant tissues. Recovery of applied excess ¹³C and ¹⁵N in plant shoots was 22% and 42%, respectively. Roots were less enriched (21 ¹³C and 277 ¹⁵N), but no attempts were made to recover roots quantitatively.     

Long-term steady-state labelling of wheat plants by use of natural 13CO2/12CO2 mixtures in an open,rapidly turned-over system

A photosynthate labelling method is presented which takes advantage of the natural difference in carbon-isotope composition () which exists between atmospheric CO₂ (-8) and commercially available compressed CO₂. Carbon dioxide with -4.0 and –27.9%., respectively, has been used for labelling. A plant growth cabinet served as the labelling compartment. CO₂-free air was continuously injected at a rate of up to 54m³·h^–1. Dilution of cabinet CO₂ by CO₂-free air was counterbalanced by addition of CO₂ with known constant . Since the labelling-cabinet atmosphere was continuously exchanged at a high rate, photosynthetic carbon-isotope discrimination was fully expressed. In order to study the distribution of carbon acquired by the plant during a defined growth period, the of CO₂ was modified by replacing, for example, atmospheric CO₂ by CO₂ with –27.9%. and the weight and 5 of plant carbon pools was monitored over time. In such an experiment the change of CO₂ was followed by a rapid change of the of sucrose in mature flag-leaf blades of wheat (Triticum aestivum L.). The 5 of sucrose stabilized near –51%., indicating complete exchange by current photosynthate. In contrast 83% of the total carbon in mature flag-leaf blades was not exchanged after 14 d continuous labelling. Differential labelling of pre- and post-anthesis photosynthate indicated that 13% of grain carbon originated from pre-anthesis photosynthesis. Carbon-isotope discrimination and its consideration in experimentation and labelling data evaluation are discussed in detail. Since the air supplied to the labelling cabinet is dry and free of CO₂, carbon-isotope discrimination and carbon turnover and partitioning can be studied over a wide range of CO₂ concentrations (0–2600 cm³ · m^–3) and vapor-pressure deficits.Abbreviation and Symbol PPFD photosynthetic photon flux density - carbon-isotope composition Dr. G. Schleser (Forschungszentrum Jülich, FRG) and Professor S. Hoernes (Mineralogisch-Petrologisches Institut, Universität Bonn) for valuable help and advice during the initial stages of the project and Professor W. Kühbauch (Institut für Pflanzenbau, Universität Bonn) for continuing support. Technical assistance of Ute Labusch, Petra Biermann, Ludwig Schmitz and Thomas Gebbing is gratefully acknowleged.

---

     

Comparisons of δ13C values in leaves of aquatic macrophytes from different habitats in Britain and Finland; some implications for photosynthetic processes in aquatic plants

Summary The ¹³C values of submerged aquatic plants from contrasting but relatively defined habitats, and the ¹³C values of emergent, floating and submerged leaves of dimorphic aquatic plants, were measured. In many instances the ¹³C values of dissolved inorganic carbon in the water were also measured. Plant ¹³C values in the vicinity of-40 to-50 were found in rapidly flowing spring waters with carbonate ¹³C values of-16 to-21, consistent with the notion that species such as Fontinalis antipyretica almost exclusively assimilate free CO₂ via RuP₂ carboxylase. Plant ¹³C values in the vicinity of-10 to-15 in sluggish water with carbonate ¹³C values of about-5 were observed, consistent with the notion that boundary layer diffusion and/or HCO₃ ^- uptake may determine the ¹³C value of submerged aquatic plants in these circumstances. Comparisons of ¹³C values of the same or related species growing in waters of similar carbonate ¹³C value but different flow rates confirmed this view; more negative ¹³C values were frequently associated with plants in fast moving water. In Britain, but not in Finland, the ¹³C values of submerged leaves of dimorphic plants were almost invariably more negative than in aerial leaves. The ¹³C value of carbonate from chalk streams and in acid springs indicate substantial inputs of respiratory CO₂, as opposed to atmospheric carbon. The contributions of these variations in ¹³C of the carbon source, and of isotope fractionation in diffusion, to the ¹³C value of submerged parts of dimorphic plants is discussed.     

Shifts in carbon isotope ratios of two C3 halophytes under natural and artificial conditions

Summary The total carbon ¹³C values of two C₃ halophytes,Salicornia europaea L. ssp.rubra (Nels.) Breitung andPuccinellia muttalliana (Schultes) Hitch., native to inland saline areas of Alberta, Canada, were determined for plants grown under controlled conditions of supplied NaCl in the nutrient solution, and for plants found growing in the field. Field specimens were collected along line transects which ran from areas of high salinity to areas of low salinity across the pattern of species zonation. The ¹³C value of the two species seemed to reflect the water potential of the soil ( _w ^soil ) as measured arbitrarily at a depth of 10 cm, becoming less negative as the _w ^soil decreased. Over a linear distance of 5.55 m,S. europaea spp.rubra showed a shift of +5.3 as the _w ^soil went from-25x10² kPa to a minimum of-73x10² kPa. ForP. nuttalliana, the ¹³C values differed by 3.4 over a distance of 7.45 m where the maximum difference in _w ^soil was 12.7x10² kPa. However, ¹³C values ofP. nuttalliana only roughly reflected the spatial trends in _w ^soil at the time of collection. In the growth chamber, the ¹³C value ofS. europaea ssp.rubra changed by a maximum of +8.0 when the solute potential of the nutrient solution ( _w ^soil ) was dropped from-0.25x10² kPa to-64.25x10² kPa; while the ¹³C value ofP. nuttalliana changed by a maximum of +10.8 when the _w ^soil was dropped from-0.25x10² kPa to-40.25x10² kPa. Linear regression analyses indicated that the ¹³C values of both species were strongly correlated (P<0.2%) with _w ^soil . The observed shifts in ¹²C may represent changes in the mode of photosynthetic CO₂ fixation. However, a number of other explanations, some of which are discussed in the text, are also possible. A proper ecophysiological interpretation of such shifts in ¹³C values of C₃ plants awaits a better understanding of the isotope fractionation mechanisms involved.     

Seasonal and diel relationships between the isotopic compositions of dissolved and particulate organic matter in freshwater ecosystems

A study of the isotopic composition of organic matter was conducted in a freshwater marsh over seasonal and diel time scales to determine the sources of dissolved organic matter (DOM) and the processes leading to its formation. Bulk C and N isotopic compositions of the bacterial fraction (0.2–0.7 m) and particulate organic matter (POM; 0.7–10 m) were compared on a seasonal basis with the change in ¹³C of DOM. The bulk isotopic data support the idea that DOM was, in part, derived from the breakdown of larger organic matter fractions. The bacterial fraction and POM were compositionally similar throughout the year, based on a comparison of the ¹³C of individual amino acids in each fraction. Annual variation in the ¹³C of amino acids in DOM was greater relative to the variation in larger fractions indicating that microbial reworking was an important factor determining the proteinaceous component of DOM. The ¹³C enrichment of serine and leucine in each organic matter fraction suggested microbial reworking was an important factor determining organic matter composition during the most productive times of year. Changes in the bulk ¹³C of DOM were more significant over daily, relative to seasonal, time scales where values ranged by 6 and followed changes in chlorophyll a concentrations. Although bulk ¹³C values for POM ranged only from –29 to –28 during the same diel period, the ¹³C of alanine in POM ranged from –30 to –22. Alanine is directly synthesized from pyruvate and is therefore a good metabolic indicator. The ¹³C of individual amino acids in DOM revealed the diel change in the importance of autotrophic versus heterotrophic activity in influencing DOM composition. Diel changes in the ¹³C of phenylalanine, synthesized by common pathways in phytoplankton and bacteria, were similar in both DOM and POM. The diel change in ¹³C of isoleucine and valine, synthesized through different pathways in phytoplankton and bacteria, were distinctly different in DOM versus POM. This disparity indicated a decoupling of the POM and DOM pools, which suggests a greater source of bacterial-derived organic matter at night. The results of this study demonstrate the use of the isotopic composition of individual amino acids in determining the importance of microbial reworking and autotrophic versus heterotrophic contributions to DOM over both diel and seasonal time scales.