Equilibrium and kinetic isotope fractionations during incomplete reactions result in minute differences in the ratio between the two stable N isotopes, 15N and 14N, in various N pools. In ecosystems such variations (usually expressed in per mil [δ15N] deviations from the standard atmospheric N2) depend on isotopic signatures of inputs and outputs, the input–output balance, N transformations and their specific isotope effects, and compartmentation of N within the system. Products along a sequence of reactions, e.g. the N mineralization–N uptake pathway, should, if fractionation factors were equal for the different reactions, become progressively depleted. However, fractionation factors vary. For example, because nitrification discriminates against 15N in the substrate more than does N mineralization, NH4+ can become isotopically heavier than the organic N from which it is derived.

Levels of isotopic enrichment depend dynamically on the stoichiometry of reactions, as well as on specific abiotic and biotic conditions. Thus, the δ15N of a specific N pool is not a constant, and δ15N of a N compound added to the system is not a conservative, unchanging tracer. This fact, together with analytical problems of measuring δ15N in small and dynamic pools of N in the soil–plant system, and the complexity of the N cycle itself (for instance the abundance of reversible reactions), limit the possibilities of making inferences based on observations of 15N abundance in one or a few pools of N in a system. Nevertheless, measurements of δ15N might offer the advantage of giving insights into the N cycle without disturbing the system by adding 15N tracer.

Such attempts require, however, that the complex factors affecting δ15N in plants be taken into account, viz. (i) the source(s) of N (soil, precipitation, NOx, NH3, N2-fixation), (ii) the depth(s) in soil from which N is taken up, (iii) the form(s) of soil-N used (organic N, NH4+, NO3−), (iv) influences of mycorrhizal symbioses and fractionations during and after N uptake by plants, and (v) interactions between these factors and plant phenology. Because of this complexity, data on δ15N can only be used alone when certain requirements are met, e.g. when a clearly discrete N source in terms of amount and isotopic signature is studied. For example, it is recommended that N in non-N2-fixing species should differ more than 5‰ from N derived by N2-fixation, and that several non-N2-fixing references are used, when data on δ15N are used to estimate N2-fixation in poorly described ecosystems.

As well as giving information on N source effects, δ15N can give insights into N cycle rates. For example, high levels of N deposition onto previously N-limited systems leads to increased nitrification, which produces 15N-enriched NH4+ and 15N-depleted NO3−. As many forest plants prefer NH4+ they become enriched in 15N in such circumstances. This change in plant δ15N will subsequently also occur in the soil surface horizon after litter-fall, and might be a useful indicator of N saturation, especially since there is usually an increase in δ15N with depth in soils of N-limited forests.

Generally, interpretation of 15N measurements requires additional independent data and modelling, and benefits from a controlled experimental setting. Modelling will be greatly assisted by the development of methods to measure the δ15N of small dynamic pools of N in soils. Direct comparisons with parallel low tracer level 15N studies will be necessary to further develop the interpretation of variations in δ15N in soil–plant systems. Another promising approach is to study ratios of 15N[ratio ]14N together with other pairs of stable isotopes, e.g. 13C[ratio ]12C or 18O[ratio ]16O, in the same ion or molecules. This approach can help to tackle the challenge of distinguishing isotopic source effects from fractionations within the system studied.

This work explored whether the natural abundances of carbon and nitrogen isotopes could be used to describe the movement of C and N within wheat plants; we also considered whether isotopic analyses of aphids or their honeydew would substitute for direct analysis of phloem exudate.

The δ13C of ears and roots (sinks) most closely matched those of the sugars+organic acids fraction (sources) in both growth stages; phloem δ13C matched that of leaf blade sugars. Xylem exudate δ13C matched no other putative (and measured) source in the ear-forming stage and matched that of whole roots and ears in the grain-filling stage. The δ15N of grain and roots (sinks) resembled that of leaf amino acids (sources) in the ear-forming stage. In the grain-filling stage, ear δ15N continued to resemble that of leaf amino acids, and δ15N of roots most closely resembled that of whole leaves. In the grain-filling stage, phloem δ15N fell between that of leaf blade amino acids and that of whole leaves and was 15N-depleted relative to internal and external NO3−-N. In both growth stages, xylem exudate δ15N was less than that of soil NO3−-N and and more than that of residual soil N after mineral N extraction. The isotopic values are generally in agreement with data from other approaches, such as isotope labelling; they show NO3−-N reduction in both shoots and roots of wheat and significant N recycling (root-shoot-phloem-root) and C movement.

Aphids might serve as a substitute for isotopic analysis of phloem δ15N, having the same value as their food source. Their excreta was 15N-enriched relative to phloem.

Developing caryopses of barley (Hordeum vulgare L. cv. Golf) were monitored for starch-branching enzyme activity for 1 month after anthesis. Homogenized caryopses from grains with the highest specific activity were used as starting material for purification of branching enzyme by FPLC chromatography. Several branching enzyme activity fractions were resolved. We have previously described the isolation of a 51/50 kDa protein with starch branching activity from one of these fractions. From three other fractions, we have now partially purified starch branching enzymes corresponding to forms I, IIa and IIb.

The occurrence of crassulacean acid metabolism (CAM) among the epiphyte flora of the lowland forest on Barro Colorado Island (BCI), Panama, was investigated. A total of 116 species was included, i.e. about 2/3 of the known epiphyte taxa. As judged from the carbon isotope ratios and the absence of Kranz anatomy, indications of CAM were found in 29 species of three families, Orchidaceae (20), Bromeliaceae (7), and Cactaceae (2). We estimate that about 25% of the epiphyte flora of BCI are CAM plants. CAM was most prevalent in exposed sites, but even in the understorey two epiphyte species engage in CAM.

Rates of carbon flux in 14 species of Antarctic bryophytes were measured under controlled conditions using an infra-red gas analysis system. The results were used to produce estimates of model parameters for respiration and photosynthesis. The relationships between respiration, photosynthesis, irradiance and temperature followed standard patterns. Temperature optima for gross and net photosynthesis were 10–20 and 0–20°C respectively, suggesting that the plants were not truly psychrophilic. Photosynthesis was saturated at 30–270 μmol m−2 s−1, consistent with the view that bryophytes are, physiologically, shade plants, although there was no evidence of photoinhibition over the range of irradiances tested (up to 700 μmol m−2 s−1). Comparison of the results with environmental data suggests that photosynthesis is usually temperature-limited during daylight in the growing season. Therefore, any change in the temperature of the habitat could affect the productivity of the bryophytes. Rates of photosynthesis varied widely between species, and these relationships were largely maintained over the range of temperatures and irradiances tested. Photosynthetic rankings were correlated with the water availability in the plant habitats, supporting the hypothesis that water is the important factor in determining the distribution of populations in Antarctic habitats.

During the middle of the 1992 austral winter in the northern Windmill Islands, continental Antarctica, a highly unusual climatic event occurred in which the air temperature exceeded 0°C for some 60 h, at the end of which there was a significant rain shower before the ambient temperature returned to subzero conditions. This event caused most of the snow cover to melt and refreeze as clear ice. Lichens were thus rehydrated in the dark, in some places completely inundated, then frozen in ice. The effect that these conditions had on the distribution of K, Na, Mg and Ca within the thallus was estimated for two of the dominant macrolichen species, Umbilicaria decussata (Vill.) Zahlbr. and Usnea sphacelata R.Br, from three sites on a knoll on Clark Peninsula. One site acted as a natural control, owing to the very deep snow cover at the site, which protected lichens from the rewetting event. Despite persistent differences between species and the various sites, there was no overall effect of the climatic event on the membrane integrity of either lichen species. Only Usnea sphacelata from the most exposed site showed a significant leakage of K across the cell membrane, which indicated a loss of membrane integrity. Overall, both species were tolerant of the extreme conditions, although Umbilicaria decussata was the more tolerant.

Monoliths of two contrasting vegetation types, a species-rich grassland on a brown earth soil over limestone and a species-poor community on a peaty gley, were transferred to solardomes and grown under ambient (350 μl l−1) and elevated (600 μl l−1) CO2 for 2 yr. Shoot biomass was unaltered but root biomass increased by 40–50% under elevated CO2. Root production was increased by elevated CO2 in the peat soil, measured both as instantaneous and cumulative rates, but only the latter measure was increased in the limestone soil. Root growth was stimulated more at 6 cm depth than at 10 cm in the limestone soil. Turnover was faster under elevated CO2 in the peat soil, but there was only a small effect on turnover in the limestone soil. Elevated CO2 reduced nitrogen concentration in roots and might have increased mycorrhizal colonization. Respiration rate was correlated with N concentration, and was therefore lower in roots grown at elevated CO2. Estimates of the C budget of the two communities, based upon root production and on net C uptake, suggest that C sequestration in the peat soil increases by c. 0·2 kg C m−2 yr−1 (=2 t ha yr−1) under elevated CO2.

A split-root growth system was used to study photosynthate partitioning to developing nodules and roots of soybean (Glycine max L., Merr.). Opposite sides of the root systems were inoculated with Bradyrhizobium japonicum at 8 and 12 d after planting (early/delayed inoculation treatment) or, alternatively, only one side was inoculated 8 d after planting (early/uninoculated treatment). Plants were incubated with 14CO2 at 24-h intervals from early inoculation until the onset of N2 fixation (acetylene reduction). After staining with Eriochrome black, root and nodule meristematic structures were excised under a dissecting microscope and their radioactivity determined by scintillation counting. The specific radioactivity of nodule structures increased with nodule development, and was as much as 4 times higher in early nodules than in roots and nodules on half-roots receiving delayed inoculation. By the time that N2 fixation could be measured in the first mature nodules, the early inoculated half-root contained over 70% of the radioactivity recovered from the entire root systems of both early/delayed and early/uninoculated treatments. These results suggest that developing nodules create a strong sink for photosynthate, and that nodules and roots compete for current photosynthate. Early initiated nodules might develop at the expense of late initiated nodules, as well as at the expense of the roots themselves.

The ultramafic sites at Meikle Kilrannoch, Scotland, have small sparsely colonized patches with soils which have highly toxic (to many non-indigenous plants) concentrations of Mg and Ni. These toxic metals are unlikely to be the main cause of the open vegetation as the indigenous plants are at least partially tolerant of them. The hypothesis that low soil nutrients were a likely cause of the open vegetation was tested by a fertilization experiment in which major nutrients and Ca were added in August 1991 and July 1992 to replicated quadrats which initially had 5·3–6·6% plant cover. There was a large increase in plant cover on major nutrient addition. The increase in rosette size, flowering, seed production, and recruitment of the dominant semelparous Cochlearia pyrenaica DC. ssp. alpina (Bab.) Dalby was studied in detail. The increased growth of Cochlearia (and some other native species at the site) on nutrient addition strengthens the argument that a major nutrient deficiency rather than a metal toxicity limits plant growth at Meikle Kilrannoch and shows that at least some stress tolerators respond with rapid growth and reproduction when nutrient limitation is removed. A small-scale experiment set up in June 1993, in which the major nutrients were added separately, suggested that P and not N, or K was the limiting element. The effects of P seem to be unrelated to any possible reduction in the availability of the toxic ions Mg 2+ and Ni2+.

Elevated [CO2] has been shown to protect photosynthesis and growth of wheat against moderately elevated [O3]. To investigate the role of ozone exclusion and detoxification in this protection, spring wheat (Triticum aestivum L. cv. Wembley) was grown from seed, in controlled-environment chambers, under reciprocal combinations of [CO2] at 350 or 700 μmol mol−1 and [O3] peaking at <5 or 60 nmol mol−1, respectively. Cumulative ozone dose to the mesophyll and antioxidant status were determined throughout flag leaf development. Catalase activity correlated with rates of photorespiration and declined in response to elevated [CO2] and/or [O3]. Superoxide dismutase activity was not significantly affected by either condition. Neither ascorbate nor glutathione content was enhanced by elevated [CO2]. In wheat, at moderately elevated [O3], our results show that stomatal exclusion plays a major role in the protective effect of elevated [CO2] against O3 damage.

The components of leghaemoglobin (Lb) from twelve different Pisum sativum L. cvs and three near-isogenic foliar mutants were investigated by anion-exchange high-performance liquid chromatography (HPLC). Five different Lb component profiles could be found. The number of components varied from four to six dependent on cultivar used. An Lb pattern composed of four Lb components could be detected in thirteen P. sativum cultivars and lines. Ten of them showed an identical profile. In nodules of each cultivar, the two known major components, LbI and LbV, but also LbIV, could be detected. Additionally, cultivar-specific Lb components could be identified, each representing up to 10% of total Lb. One of these components, LbIII, has been described previously, but three new Lb components (LbII, LbVI, and LbVII) were found. The presence of all Lb components detected by HPLC was confirmed by analytical isoelectric focusing. Further, it was shown that age-dependent changes in the relative concentrations of LbI and LbV are common in P. sativum and that these variations are independent of breeding lines and cultivars.

The growth of the grass Brachypodium pinnatum (L.) Beauv. in Dutch nutrient-poor chalk grasslands increases with enhanced nitrogen supply, whereas other grass species also require an enhanced phosphorus supply for a similar response (e.g. Dactylis glomerata L.), or are competitively suppressed at any increase in nutrient supply (e.g. Briza media L.). We investigated whether this interspecific variation in response to N and P supply is caused by differences in P productivity (PP), i.e. the instantaneous rate of biomass production per unit of P present in the plant. We hypothesized that PP is highest in Brachypodium pinnatum, in contrast to N productivity which is known to be the highest in Dactylis glomerata. Phosphorus productivity and its components were studied using a growth analysis with four exponential P addition rates of 0·03, 0·06, 0·09 and 0·11/0·15 mg P mg−1 P d−1.

Although Brachypodium pinnatum allocated more P to its leaf blades, it had a lower P productivity at high N and low P supply than did Dactylis glomerata. This was associated with a higher productivity per unit leaf P in Dactylis glomerata. Across all species and treatments, leaf PP showed a distinct negative correlation with P concentration per leaf area, regardless whether the variation in area-based leaf P concentration was caused by variation in leaf thickness, leaf tissue mass density or mass-based P concentration. A possible explanation for this would be a positive correlation between leaf chlorophyll concentration and P concentration, leading at high concentrations to shading within the leaf and to a low photosynthetic rate per unit leaf P. We conclude that a high PP is determined by the ability of a plant to distribute its P over a large leaf area, rather than by greater allocation of P to the leaves. Interspecific relationships for P productivity are similar to those known for N productivity.

Nine different clones of six species of Salix (Salix cordata Muhlenb. non Michaux, S. fragilis L., S. caprea L., S. cinerea L., S. burjatica Nazarov. and S. viminalis L.) and one hybrid (S.×calodendron Wimm.) were exposed to heavy metals in solution culture in an attempt to increase innate metal resistance. Resistance was estimated using comparative root measurements, and metal uptake was also studied. The first experiment entailed pretreatments with background nutrient solution, or 0·25 and 0·50 mg Cu l−1 amendments, and re-exposure to each of the same concentrations. In a second experiment clones were exposed to sub-toxic concentrations of single metals (0·15 mg Cu l−1, 0·15 mg Cd l−1 or 2·5 mg Zn l−1) and dual-combination treatments (0·075 mg Cu l−1+0·075 mg Cd l−1, 0·075 mg Cu l−1+1·25 mg Zn l−1 or 0·075 mg Cd l−1+1·25 mg Zn l−1)1 with concentrations gradually raised 10-fold over 128 d. Plants tested in the first experiment, following pre-exposure to Cu, were no more resistant to subsequent exposure to this metal. In the second experiment, gradual cumulative doses resulted in reduced phytotoxicity and increased resistance, most notably to Cd. There appeared to be an inverse relationship between metal uptake and resistance. Copper uptake was restricted to the roots, whereas Cd and Zn were more evenly distributed throughout the plant. Exposure to dual combinations of metals resulted in several interaction effects on uptake: increased root-bound Cu in all combinations, and the increase in uptake of both Cd and Zn into the root tissues when supplied with Cu. The implications of these results for the use of willows in phytoremediation programmes are discussed.

A common feature of many pollutant exposure studies is that data from these experiments often consist of either plant biomass or yield, and the response to the pollutant is usually based on a simple comparison of means determined at the end of each growing season. This type of data is now being used to quantify critical levels for different types of vegetation. Such an approach is, however, inappropriate for the relatively short-term exposure studies with trees since, due to their longevity, it is not possible to determine a final yield. Instead these studies should be regarded as a type of intervention experiment in which only a small part of the life cycle of the tree is investigated. Moreover, critical levels as they are now defined focus on the cumulative exposure to ozone concentrations over time. Hence, any analysis of the effects of ozone on the growth of trees should similarly focus on the behaviour of growth functions over time and not on a comparison of biomass at the end of a growing season or experiment.

Here we report on the statistical analysis of a longitudinal study, where the term ‘longitudinal’ refers to the analysis of repeated measurements over time, and which was used to investigate relative differences in the growth of Sitka spruce and Norway spruce seedlings during three summers' exposure to ozone over three (Norway) or four (Sitka) growing seasons. Measurements of total seedling height and stem diameter were made at frequent intervals over the period of the experiment and the above-ground growth of individual trees (as log d2h) analysed for each growing season using a statistical model of the form: m(t)=a+b(t−t0). No statistically significant differences in the growth of Norway spruce were observed after three summers of ozone exposure. The growth of Sitka spruce was, however, reduced by ozone during the third growing season and in the following year, even in the absence of the pollutant.

For the Sitka spruce, the fitted model was used to calculate the time at which a 10% reduction in growth had occurred in the ozone-exposed trees. In combination with ozone AOT40 indices for the relevant growing seasons, this was then used to determine a critical level of 21·3 ppm-h for this species. Since the growth of the Norway spruce was unaffected by exposure to ozone it was not possible to calculate a critical level other than to surmise that it is in excess of 30 ppm-h.

Genetic diversity, population genetic structure and gene flow in Calluna vulgaris (L.) Hull were assessed by means of seven allozyme loci scored in 18 populations from the South-Western area of the species' range. Genetic diversity was lower (HT=0·20) than reported for long-lived widespread species but was characterized by a high number of alleles per locus (5·60 at the species level) of which more than 70% were rare. More than 95% of genetic variation was found at the intrapopulation level (GST=0·047). High levels of past gene flow were inferred, based on the allozyme data (Nm=5·2 from GST, Nm=10·2 from the ‘private allele’ method). Calluna vulgaris exhibited several geographic patterns of genetic variation. Both cluster analysis, constructed with various genetic distances and principal components analysis showed that Spanish and Pyrenean populations were clearly different from those collected in the Massif Central and Belgium. Also, a trend for decreasing genetic diversity towards Northern populations was detected. These patterns might be related to the post-glacial history of Calluna. In addition, it is shown that isolation by distance has played a role in the geographic shaping of genetic variation in this species.

Many hypotheses have been developed to explain the adaptive nature of insect galls. One of these, the nutrition hypothesis, states that gall formers have advantages over other insects because gall tissue provides a better (higher quality) food source than unmodified tissue. However, this has rarely been experimentally tested. In a test of this hypothesis, we grew plants of Cirsium arvense (L.) Scop. in a factorial design with two main treatments: the addition of nitrogen (to enhance foliar N levels) and of fungicide (to reduce colonization of roots by arbuscular mycorrhizal fungi). Mycorrhizal fungi have been shown previously to reduce the N concentration of host plants. Plants were exposed to adult gall flies, Urophora cardui L., and maintained through one season to allow maturation of galls.

Reduction of the percentage mycorrhizal colonization by fungicide resulted in an elevation of total stem N comparable to that achieved by N addition, but gall N concentration remained unchanged in all treatments. Nitrogen application elevated stem N levels when mycorrhizal fungi were present, but application of both compounds together did not result in any increase over either single treatment. Fungicide application resulted in larger galls, which contained more larval chambers, with more live, and heavier, larvae. However, the main effects of N were not significant, as N addition only increased fly performance on plants where mycorrhizas were not reduced.

It is suggested that U. cardui gall inhabitants can manipulate N at an optimal level and thus might conform to a modified version of the nutrition hypothesis. Mycorrhizal colonization might reduce gall fly performance by delaying the appearance, or impairing the quality, of secondary nutritive tissue in the gall. Future tests of the nutrition hypothesis should include a consideration of the plant's mycorrhizal status.

The ability of ryegrass (Lolium perenne L.) to take up and utilize aspartic acid (Asp) and serine (Ser), and the effect of colonization of the roots by the arbuscular mycorrhizal (AM) fungus Glomus fasciculatum (Thax. sensu Gerd.) were studied. The seedlings were grown under controlled conditions in a series of micro-lysimeters. All plants were fed with a nutrient solution containing either nitrate, Asp or Ser as the sole N source. After 49 d, they were supplied with 15N labelled nitrate, Asp or Ser for 1 h and harvested.

AM colonization increased the growth and total N content of the plants in all cases. Similarly, the amount of Asp or Ser taken up was higher in AM than in control plants. There were no differences in biomass production between the nitrate and Ser-fed plants. However uptake rates were lower for Ser than for nitrate. Growth of the Asp-fed plants was significantly less than the other two treatments, and uptake of 15N-Asp was lower than uptake of 15N-Ser. Analysis of 15N incorporation into the amino acids extracted from the roots suggests the hydrolysis of Ser followed by re-assimilation of the resulting ammonia via the GS-GOGAT cycle. There were no differences in the patterns of accumulation of amino acids in the root-zone of control and AM-ryegrass. The implication of these results for the pathway of nitrogen transfer between plants is discussed.

The amounts of cyclic AMP (cAMP), fructose-2,6-bisphosphate (F26BP), trehalose and glycogen were determined in cell suspension cultures of the ectomycorrhiza-forming fungus Amanita muscaria (L. ex Fr.) Hooker. For the assay of cAMP a protocol was developed that enabled the detection of as little as 50 fmol of this secondary messenger by an enzyme-linked immuno assay (EIA). Values varied from <1 and up to 5 pmol cAMP mg−1 d. wt according to the age of the fungal culture. Typically, a transient increase in cAMP occurred after c. 4 d of culture of the fungus on glucose-containing medium. This increase (up to 100%) was followed by the start of the logarithmic growth phase, and by a more persistent increase in F26BP. In parallel, glucose in the medium started to decrease, whilst the amounts of fungal carbohydrates, especially the disaccharide trehalose, increased. From these data we assume that a high initial rate of glucose uptake caused an increase in the fungal pools of storage carbohydrates and, via activation of an adenylate cyclase, of cAMP. According to data reported for yeast cells this should enhance the formation of F26BP by phosphorylation of relevant enzymes. In animal and yeast cells an increase in the concentration of F26BP stimulates glycolysis by activation of the ATP-dependent phosphofructokinase (PFK). A. muscaria also possesses an F26BP activated PFK and, under conditions of symbiosis, host-derived carbohydrates are supplied mainly in the form of glucose. The implications of these findings to the regulation of carbohydrate metabolism of symbiotic plant root/fungus structures (ectomycorrhiza) are discussed.

The identity of black alder (Alnus glutinosa (L.) Gaertn.) ectomycorrhizas was investigated using PCR/RFLP analysis of the ITS region from 16 morphotypes sampled at a 60-yr-old black alder stand. A comparison was made with restriction patterns from sporocarps of 28 mycobionts, of which 16 originated from the same stand, the remaining 12 came from two geographically distant alder stands. Eight of the mycorrhizal types could thus be identified, whereas eight mycorrhizal types remained unidentified. The identified mycorrhizas belonged to the genera Russula, Lactarius, Naucoria and Cortinarius. Four of the identified ectomycorrhizal types had identical PCR/RFLP profiles to corresponding fruit bodies from all investigated stands with no detectable intraspecific variation, despite the geographical distance of c. 300 km between the sampling locations. By contrast, intraspecific variation between sporocarps from the different locations was detected in Paxillus rubicundulus, mycorrhizas of which were not found. The diversity of fruiting alder mycobionts at the main experimental plot only partly matched the diversity observed from mycorrhizas when comparing their PCR/RFLP profiles. The results are discussed regarding sampling techniques, PCR/RFLP analyses and ecological aspects.