Beneficial effects of sustained activity on the use of dietary protein and carbohydrate traced with stable isotopes 15N and 13C in gilthead sea bream (Sparus aurata)

To determine the effects of sustained swimming on the use and fate of dietary nutrients in gilthead sea bream, a group of fish were forced to undertake moderate and sustained swimming (1.5 BL s^?1) for 3 weeks and compared with a control group undertaking voluntary activity. The exercise group showed a significant increase in specific growth rate (C: 1.13 ± 0.05; E: 1.32 ± 0.06 % day^?1, P < 0.05) with no significant change in food intake (C: 3.56 ± 0.20; E: 3.84 ± 0.03 % of body weight). The addition of ¹³C-starch and ¹⁵N-protein to a single meal of 1 % ration allowed analysis of the fate of both nutrients in several tissues and in their components, 6 and 24 h after force-feeding. In exercised fish improved redistribution of dietary components increased the use of carbohydrates and lipid as fuels. Gilthead sea bream have a considerable capacity for carbohydrate absorption irrespective of swimming conditions, but in trained fish ¹³C rose in all liver fractions with no changes in store contents. This implies higher nutrient turnover with exercise. Higher retention of dietary protein (higher ¹⁵N uptake into white muscle during the entire post-prandial period) was found under sustained exercise, highlighting the protein-sparing effect. The combined effects of a carbohydrate-rich, low-protein diet plus sustained swimming enhanced amino acid retention and also prevented excessive lipid deposition in gilthead sea bream.     

The trophic structure of bark-living oribatid mite communities analysed with stable isotopes (15N, 13C) indicates strong niche differentiation

The aim of the present study was to identify food sources of bark-living oribatid mites to investigate if trophic niche differentiation contributes to the diversity of bark living Oribatida. We measured the natural variation in stable isotope ratios (¹⁵N/¹⁴N, ¹³C/¹²C) in oribatid mites from the bark of oak (Quercus robur), beech (Fagus sylvatica), spruce (Picea abies) and pine (Pinus sylvestris) trees and their potential food sources, i.e., the covering vegetation of the bark (bryophytes, lichens, algae, fungi). As a baseline for calibration the stable isotope signatures of the bark of the four tree species were measured and set to zero. Oribatid mite stable isotope ratios spanned over a range of about 13 δ units for ¹⁵N and about 7 δ units for ¹³C suggesting that they span over about three trophic levels. Different stable isotope signatures indicate that bark living oribatid mites feed on different food sources, i.e., occupy distinct trophic niches. After calibration stable isotope signatures of respective oribatid mite species of the four tree species were similar indicating close association of oribatid mites with the corticolous cover as food source. Overall, the results support the hypothesis that trophic niche differentiation of bark living oribatid mites contributes to the high diversity of the group.     

Use of stable isotopes to quantify nitrogen, potassium and magnesium dynamics in young Scots pine (Pinus sylvestris)

Two-yr-old Scots pine ( Pinus sylvestris ) seedlings were grown in sand culture for 1 yr with a generous supply of a balanced nutrient solution. Trees were repotted into clean sand in February 1998 and given either a reduced or adequate nutrient supply containing enriched ¹⁵N, ⁴¹K and ²⁶Mg to label nutrient uptake during spring 1998. Trees doubled their biomass during the experiment. Whole-tree net photosynthesis was reduced by 43% after 95 d in trees that received the lower nutrient supply ( P < 0.001), although differences in biomass between the two treatments were less pronounced. Remobilization contributed 83, 82 and 52% of the N, K and Mg, respectively, used to support growth of new tissues in trees that received reduced nutrient supply. Those receiving the higher nutrient supply still obtained 44–59% of nutrients used for spring growth of new tissues from remobilization. Current nutrient supply had no significant effect on the amount of N or Mg remobilized to new tissues but K remobilization was less in trees that received the lower nutrient supply ( P = 0.025). The importance of remobilization in young trees and problems associated with quantifying internal cycling of nutrients are discussed.     

Photoreactivation of the UV light effects on growth of Scots pine (Pinus sylvestris L.) seedlings

Summary Ultraviolet-B light (UV-B) and ultraviolet-A light (UV-A) at higher doses exert a strong inhibitory (toxic) effect on axis growth in Scots pine (Pinus sylvestris L.) seedlings. This effect is unrelated to control of growth rate by phytochrome. Rather, after a toxic UV dose growth of the pine seedling no longer responded to phytochrome. Both, the effect of UV-B as well as the inhibiting effect of UV-A could be photoreactivated by blue light (B). The action of UV-A was 2 fold: (i) it exerted a toxic effect which could be photoreactivated by B, and (ii) applied after UV-B it photoreactivated to some extent the toxic UV-B effect. Obviously, the UV-A range causes a toxic effect, and at the same time is capable of photoreactivating the toxic UV effect. At higher doses the toxic effect prevails.     

Labelling halophilic Archaea using 13C and 15N stable isotopes: a potential tool to investigate haloarchaea consumption by metazoans

The use of stable isotope (SI) labelling and tracing of live diets is currently considered one of the most comprehensive tools to detect their uptake and assimilation by aquatic organisms. These techniques are indeed widely used in nutritional studies to follow the fate of specific microbial dietary components, unraveling trophic interactions. Nevertheless, to the current date our understanding of aquatic trophic relationships has yet to include a whole domain of life, the Archaea. The aim of the present research was, therefore, to describe a halophilic Archaea (haloarchaea) labelling procedure, using the SI ¹³C and ¹⁵N, to enable the application of SI tracing in future studies of haloarchaea consumption by aquatic metazoans. To this end, three ¹³C enriched carbon sources and two ¹⁵N enriched nitrogen sources were tested as potential labels to enrich cells of three haloarchaea strains when supplemented to the culture medium. Our overall results indicate ¹³C-glycerol as the most effective carbon source to achieve an efficient ¹³C enrichment in haloarchaea cells, with Δδ¹³C values above 5000‰ in all tested haloarchaea strains. As for ¹⁵N enriched nitrogen sources, both (¹⁵NH₄)₂SO₄ and ¹⁵NH₄Cl seem to be readily assimilated, also resulting in efficient ¹⁵N enrichment in haloarchaea cells, with Δδ¹⁵N values higher than 20,000‰. We believe that the proposed methodology will allow for the use of SI labelled haloarchaea biomass in feeding tests, potentially providing unambiguous confirmation of the assimilation of haloarchaea biomass by aquatic metazoans.

     

Long term effect of liming and fertilization on ectomycorrhizal colonization and tree growth in old Scots pine (Pinus sylvestris L.) stands

In this study, we surveyed the long term effects of liming and fertilizing in old Scots pine stands on the ectomycorrhiza (ECM) colonization, tree growth and needle nutrient concentration 35 years later. Four mature stands of Scots pine on low productive mineral soil were limed in 1959 and 1964 with total doses of limestone ranging from 3 to 15 Mg ha^?1 and fertilized with nitrogen (N) in 1970. Thirty-five years after the first liming treatment, all stands were analysed for tree growth and needle nutrient concentrations and two of the stands were also analysed for ECM colonization. ECM colonization increased significantly with liming from 61.5% in the control plots to 88% in the plot with the highest limestone dose. ECM colonization increased with increasing pH in the humus layer from 62% colonization at pH?=?3.5 to 90% at pH?=?6.5 and decreased with increasing amount of extractable phosphorus (P) in the humus. Liming did not affect the frequencies of different ECM morphotypes or dead short root tips, the fine root biomass or necromass. ECM colonization was uncorrelated with needle nutrient concentrations or tree increment. Liming did not significantly affect tree growth. However, nutrient concentrations of current-year needles were affected by prior liming. Ca concentrations in current-year needles increased from approximately 15 mg g^?1 in control treatments to more than 30 mg g^?1 in limed plots, whereas concentrations of Mn, Al, Fe, and in two stands, B, decreased due to liming. In conclusion, liming with doses up to 15 Mg ha^?1 was detectable in stands 35 years after treatment. The liming significantly increased the ECM colonization of Scots pine fine roots, increased the needle nutrient concentration of Ca and decreased the needle concentrations of Mn, Al, and Fe.     

The effects of UV-B radiation on epidermal anatomy in loblolly pine (Pinus taeda L.) and Scots pine (Pinus sylvestris L.)

The effects of enhanced UV‐B radiation on the needle anatomy of loblolly pine (Pinus taeda L.) and Scots pine (Pinus sylvestris L.) were studied in the field under supplemental UV‐B radiation supplied by a modulated irradiation system. The supplemental UV‐B levels were designed to simulate either a 16 or 25% loss of stratospheric ozone over College Park, Maryland. Enhanced UV‐B radiation caused different responses in these two species. The needles of loblolly pine had larger amounts of tannin in the lumen of epidermal cells and more wall‐bound phenolics in the outer epidermal walls of UV‐B‐treated needles, whereas the most pronounced effect on Scots pine needles was increased cutinization. In both species, the outer epidermal cell walls thickened and the needle cross‐sectional and mesophyll areas decreased (statistically significantly only in Scots pine). This suggests that more carbon may have been allocated to the protection mechanisms at the expense of photosynthetic area. The difference in response between these species suggests that the response to UV‐B radiation is not mediated by a single mechanism and that no generalization with regard to the effects of UV‐B on conifers can be made.     

A study of root distribution and growth in a Pinus sylvestris L. (Scots pine) plantation in east anglia

Summary The root systems of Scots pine in a plantation were studied by three methods; soil coring, soil monoliths, and a root trench with observation windows were used to estimate root length, root diameters and the initiatio of new root tips. The vertical and horizontal distribution of roots is described and root distribution has been related to distance from the tree and soil heterogeneity. It was found that the initiation of new root tips was not readily relatable to the soil environment and the usefulness of the root window technique for observing new root tips is questioned.     

Biosynthetic uniform 13C,15N-labelling of zervamicin IIB. Complete 13C and 15N NMR assignment.

Zervamicin IIB is a member of the alpha-aminoisobutyric acid containing peptaibol antibiotics. A new procedure for the biosynthetic preparation of the uniformly 13C- and 15N-enriched peptaibol is described This compound was isolated from the biomass of the fungus-producer Emericellopsis salmosynnemata strain 336 IMI 58330 obtained upon cultivation in the totally 13C, 15N-labelled complete medium. To prepare such a medium the autolysed biomass and the exopolysaccharides of the obligate methylotrophic bacterium Methylobacillus flagellatus KT were used. This microorganism was grown in totally 13C, 15N-labelled minimal medium containing 13C-methanol and 15N-ammonium chloride as the only carbon and nitrogen sources. Preliminary NMR spectroscopic analysis indicated a high extent of isotope incorporation (> 90%) and led to the complete 13C- and 15N-NMR assignment including the stereospecific assignment of Aib residues methyl groups. The observed pattern of the structurally important secondary chemical shifts of 1H(alpha), 13C=O and 13C(alpha) agrees well with the previously determined structure of zervamicin IIB in methanol solution.     

Daily dynamics in xylem cell radial growth of Scots pine (Pinus sylvestris L.)

Daily dynamics of radial cell expansion during wood formation within the stems of 25-year-old Scots pine trees (Pinus sylvestris L.), growing in field conditions, were studied. The samples of forming wood layers were extracted 4 times per day for 3 days. Possible variations in the growth on different sides of the stem, duration of cell development in radial cell expansion phase and dynamics of cell growth in this phase were taken into account. The perimeters of tracheid cross-sections as a reflection of primary cell wall growth were the criterion of growth in a radial direction. For the evaluation of growing cell perimeters a special system for digital processing and image analysis of tracheid cross-sections of the forming wood was used. Growth rate for certain time intervals was estimated by the change in the relation of the perimeter of each observed cell in each of ten tracheid rows in each of 12 trees to the perimeter of the xylem cell of the same row before the expansion. Temporal differences in average values of the relations were estimated by Analyses of Variance. The existence of daily dynamics of Scots pine xylem cell radial growth has been proved. Intensive growth of pine tracheids has been shown to occur at any time of the day and to depend on the temperature regime of the day and the night as well as water supply of stem tissues. Moreover, reliable differences (P = 0.95) in the increment of cell walls during tracheid radial expansion have been found. Pulsing changes of the water potentials both of the cell and the apoplast, as the reason for the fluctuations of radial cell growth rate, were discussed.     

Plastids and glycolipids in the stemwood of Pinus sylvestris L.

Summary The ultrastructure of plastids in xylem ray parenchyma cells of Pinus sylvestris L. was studied and compared with the glycolipid composition of the stemwood. Seasonal changes of the ultrastructure were studied by taking samples regularly throughout the year. The plastids resemble amyloplasts. They usually have one large starch grain, and considerable variation in structure and starch content was observed, especially in the innermost sapwood and in the sapwood-heartwood transition zone. Electron-dense deposits were observed attached to the plastid membranes and envelopes, especially in the transition zone, from April to November. The plastids were aggregated near the nucleus and the starch disappeared during the winter (January–March). The glycolipids, monogalactosyldiacylglycerol (MGDG) and di-galactosyldiacylglycerol (DGDG), were present only in the sapwood, in trace amounts. The glycolipid content was slightly greater in the outer sapwood than in the sapwood-heartwood transition zone. DGDG was the dominant lipid of the two.     

Using stable isotope natural abundances (delta 15N and delta 13C) to integrate the stress responses of wild barley (Hordeum spontaneum C. Koch.) genotypes

To integrate the complex physiological responses of plants tostress, natural abundances (     

Long-term effects of boron and copper on phenolics and monoterpenes in Scots pine (Pinus sylvestris L.) needles

Backgroud and aims

Plant boron (B) status is known to affect plant secondary metabolites but most studies have been short termed and in controlled environments. Copper (Cu) effects on phenolics are better known at toxic than at low levels. Here, the chemistry of Scots pine (Pinus sylvestris L.) needles was studied 20 years after fertilisation with B and Cu in a long-term field experiment on a drained boreal peatland.

Methods

Phenolic compounds were analysed from three needle year classes using high performance liquid chromatography (HPLC) and condensed tannins with modified acid-butanol assay. Monoterpenes in the youngest needles were analysed by gas chromatography–mass spectrometry (GC–MS).

Results

Needle B concentrations were at deficient level in controls (5.7 μg g^?1), but at the optimum level (12 μg g^?1) still 20 years after fertilisation. Copper concentrations were low but not deficient (4.0 μg g^?1 in unfertilised, 4.8 μg g^?1 in fertilised). Needle ageing increased the concentrations of individual phenolics in most cases, but decreased the concentration of condensed tannins. The concentrations of several individual phenolics were reduced by B fertilisation compared to B-deficient control, significantly in the cases of (+)-catechin and a neolignan. The concentrations of eight compounds and the sum of small-molecule phenolics were higher in Cu fertilised trees. Condensed tannins and monoterpenes were not affected by the micronutrients.

Conclusions

Boron and copper additions affected mostly the same phenolic compounds, but B decreased while Cu increased their concentrations, Cu effects being clearer. The higher phenolic concentrations in B deficient trees were not likely large enough to explain leader dieback in B-deficient trees. The effects and interactions of these micronutrients need to be further studied in field conditions to establish firstly if the changes in phenolics are consistent among species, and secondly what mechanisms lead to the changes. Although small, the changes in phenolic concentrations may affect the interactions of the trees with their biotic and abiotic environment, when consistent over many years.     

A novel approach for uniform (13)C and (15)N labeling of DNA for NMR studies.

A novel method is proposed for large-scale synthesis of (13)C- and (15)N-labeled DNA for NMR studies. In this methodology, endonuclease-sensitive repeat amplification (ESRA), a modified PCR strategy, has been used to amplify tandem repeats of the target DNA sequence. The design of the template is such that restriction enzyme (RE) sites separate repeats of the target sequence. The ESRA product is then cloned into a suitable vector. The Escherichia coli cells harboring the plasmid are grown in minimal medium containing [(13)C]glucose and (15)NH(4)Cl as the sole source of carbon and nitrogen, respectively. The target sequence is released by RE digestion of the plasmid, followed by purification using PAGE. Under optimized conditions, the yield ( approximately 5 mg/liter of culture) of (13)C/(15)N-labeled DNA prepared using this approach is found to be several times higher compared to other known enzymatic methods. Successful incorporation of the isotopes has been confirmed using 2D NMR techniques.     

Dormancy release in Pinus sylvestris L. and Picea abies (L.) Karst. seedlings: effects of intermittent warm periods during chilling

Summary Experiments designed to test three simulation models were used to study the effects of intermittent warm periods during the chilling period on dormancy release in 2-year-old seedlings of Pinus sylvestris L. and Picea abies (L.) Karst. The effect of the intermittent period varied according to its timing. Compared with corresponding continuous chilling treatments, the intermittent periods (1) after 1–3 weeks of chilling increased the proportion of the seedlings for which dormancy was subsequently released, and (2) after 4–7 weeks of chilling substantially diminished this proportion. The intermittent periods did not affect the time required for growth initiation in forcing conditions. These results support a simulation model with a strict end-point for the rest period. On the basis of the experimental results, division of the dormant period into three sub-periods is proposed.     

Topographic influences on radial growth of Scots pine (Pinus sylvestris L.) at small spatial scales

Dendroecological and numerical methods were used to study the influence of topographic position on radial growth of Scots pine (Pinus sylvestris) stands exposed to soil dryness. The correlation structure of total tree-ring width and latewood width of eight scattered populations representing various topographic habitats (steep south-facing slopes, plateaus and hollows) within a rock-slide area (750 m a.s.l.) of about 1 km² was investigated by principal component analysis. Scatter plots of component loadings indicated that (i) total ring width and latewood width are influenced by various climatic factors, (ii) stands growing at similar topographic position show a high agreement in year-to-year variability of radial growth, and (iii) distinct effects of topographic features (slope aspect, slope magnitude) on tree growth are modified by local disturbances (erosion, grazing) and the age structure of stands. Furthermore, both the time series of component scores and non-metric multidimensional scaling of chronologies indicated years where extremely limiting or favorable climate conditions prevailed throughout the study area (pointer years). The influence of climate on tree growth in various topographic habitats was mediated through the influence of climatically stressful years. Because stands are located at sites with different levels of water stress, growth differences between chronologies are considered to be caused by site-specific susceptibility of tree growth to soil dryness. Significant correlations between precipitation in April to June and ring-width confirm that water availability is the primary growth-limiting factor within the study area. These small-scale variations in growth-climate relationships have significant implications for dendroclimatological studies. So paleoclimatic reconstructions based on tree rings will have to assure that an unbiased data set is used, which compensates for local growth-variabilities due to site related environmental stresses.     

CO_H(N)CACB experiments for assigningbackbone resonances in 13C/15N-labeledproteins

A triple resonance NMR experiment, denoted CO_H(N)CACB, correlates¹H^N and ¹³CO spins with the¹³C and¹³C spins of adjacent amino acids. Thepulse sequence is an out-and-back design that starts with¹H^N magnetization and transfers coherence viathe ¹⁵N spin simultaneously to the ¹³CO and¹³C spins, followed by transfer to the¹³C spin. Two versions of the sequence arepresented: one in which the ¹³CO spins are frequency labeledduring an incremented t₁ evolution period prior to transfer ofmagnetization from the ¹³C to the¹³C resonances, and one in which the¹³CO spins are frequency labeled in a constant-time mannerduring the coherence transfer to and from the¹³C resonances. Because ¹³COand ¹⁵N chemical shifts are largely uncorrelated, thetechnique will be especially useful when degeneracy in the¹H^N-¹⁵N chemical shifts hindersresonance assignment. The CO_H(N)CACB experiment is demonstrated usinguniformly ¹³C/¹⁵N-labeled ubiquitin.