Search

The present study explores the interaction of water supply and rhizobia inoculation on CO

We used instantaneous temperature responses of CO2 -respiration to explore temperature acclimation dynamics for Eucalyptus grandis grown with differing nitrogen supply. A reduction in ambient temperature from 23 to 19 °C reduced light-saturated photosynthesis by 25% but increased respiratory capacity by 30%. Changes in respiratory capacity were not reversed after temperatures were subsequently increased to 27 °C. Temperature sensitivity of respiration measured at prevalent ambient temperature varied little between temperature treatments but was significantly reduced from ~105 kJ mol-1 when supply of N was weak, to ~70 kJ mol-1 when it was strong. Temperature sensitivity of respiration measured across a broader temperature range (20-40 °C) could be fully described by 2 exponent parameters of an Arrhenius-type model (i.e., activation energy of respiration at low reference temperature and a parameter describing the temperature dependence of activation energy). These 2 parameters were strongly correlated, statistically explaining 74% of observed variation. Residual variation was linked to treatment-induced changes in respiration at low reference temperature or respiratory capacity. Leaf contents of starch and soluble sugars suggest that respiratory capacity varies with source-sink imbalances in carbohydrate utilization, which in combination with shifts in carbon-flux mode, serve to maintain homeostasis of respiratory temperature sensitivity at prevalent growth temperature.

Phytopathogenic fungi infections induce plant defence responses that mediate changes in metabolic and signalling processes with severe consequences for plant growth and development. Sphaeropsis tip blight, induced by the endophytic fungus Sphaeropsis sapinea that spreads from stem tissues to the needles, is the most widespread disease of conifer forests causing dramatic economic losses. However, metabolic consequences of this disease on bark and wood tissues of its host are largely unexplored. Here, we show that diseased host pines experience tissue dehydration in both bark and wood. Increased cytokinin and declined indole-3-acetic acid levels were observed in both tissues and increased jasmonic acid and abscisic acid levels exclusively in the wood. Increased lignin contents at the expense of holo-cellulose with declined structural biomass of the wood reflect cell wall fortification by S. sapinea infection. These changes are consistent with H2 O2 accumulation in the wood, required for lignin polymerization. Accumulation of H2 O2 was associated with more oxidized redox states of glutathione and ascorbate pools. These findings indicate that S. sapinea affects both phytohormone signalling and the antioxidative defence system in stem tissues of its pine host during the infection process.

Amino acids represent an important component in the diet of the Venus flytrap (Dionaea muscipula), and supply plants with much needed nitrogen resources upon capture of insect prey. Little is known about the significance of prey-derived carbon backbones of amino acids for the success of Dionaea's carnivorous life-style. The present study aimed at characterizing the metabolic fate of 15 N and 13 C in amino acids acquired from double-labeled insect powder. We tracked changes in plant amino acid pools and their δ13 C- and δ15 N-signatures over a period of five weeks after feeding, as affected by contrasting feeding intensity and tissue type (i.e., fed and non-fed traps and attached petioles of Dionaea). Isotope signatures (i.e., δ13 C and δ15 N) of plant amino acid pools were strongly correlated, explaining 60% of observed variation. Residual variation was related to contrasting effects of tissue type, feeding intensity and elapsed time since feeding. Synthesis of nitrogen-rich transport compounds (i.e., amides) during peak time of prey digestion increased 15 N- relative to 13 C- abundances in amino acid pools. After completion of prey digestion, 13 C in amino acid pools was progressively exchanged for newly fixed 12 C. The latter process was most evident for non-fed traps and attached petioles of plants that had received ample insect powder. We argue that prey-derived amino acids contribute to respiratory energy gain and loss of 13 CO2 during conversion into transport compounds (i.e., 2 days after feeding), and that amino-nitrogen helps boost photosynthetic carbon gain later on (i.e., 5 weeks after feeding).

The present study was performed to elucidate the fate of carbon (C) and nitrogen (N) derived from protein of prey caught by carnivorous Dionaea muscipula. For this, traps were fed 13 C/15 N-glutamine (Gln). The release of 13 CO2 was continuously monitored by isotope ratio infrared spectrometry. After 46 h, the allocation of C and N label into different organs was determined and tissues were subjected to metabolome, proteome and transcriptome analyses. Nitrogen of Gln fed was already separated from its C skeleton in the decomposing fluid secreted by the traps. Most of the Gln-C and Gln-N recovered inside plants were localized in fed traps. Among nonfed organs, traps were a stronger sink for Gln-C compared to Gln-N, and roots were a stronger sink for Gln-N compared to Gln-C. A significant amount of the Gln-C was respired as indicated by 13 C-CO2 emission, enhanced levels of metabolites of respiratory Gln degradation and increased abundance of proteins of respiratory processes. Transcription analyses revealed constitutive expression of enzymes involved in Gln metabolism in traps. It appears that prey not only provides building blocks of cellular constituents of carnivorous Dionaea muscipula, but also is used for energy generation by respiratory amino acid degradation.

We studied acclimation of leaf gas exchange to differing seasonal climate and soil water availability in slow-growing date palm (Phoenix dactylifera) seedlings. We used an extended Arrhenius equation to describe instantaneous temperature responses of leaf net photosynthesis (A) and stomatal conductance (G), and derived physiological parameters suitable for characterization of acclimation (T-opt, A(opt) and T-equ). Optimum temperature of A (T-opt) ranged between 20-33 degrees C in winter and 28-45 degrees C in summer. Growth temperature (T-growth) explained c. 50% of the variation in T-opt, which additionally depended on leaf water status at the time of measurement. During water stress, light-saturated rates of A at T-opt (i.e. A(opt)) were reduced to 30-80% of control levels, albeit not limited by CO2 supply per se. Equilibrium temperature (T-equ), around which A/G and substomatal [CO2] are constant, remained tightly coupled with T-opt. Our results suggest that acclimatory shifts in T-opt and A(opt) reflect a balance between maximization of photosynthesis and minimization of the risk of metabolic perturbations caused by imbalances in cellular [CO2]. This novel perspective on acclimation of leaf gas exchange is compatible with optimization theory, and might help to elucidate other acclimation and growth strategies in species adapted to differing climates.

Here, we characterized nitrogen (N) uptake of beech (Fagus sylvatica) and their associated ectomycorrhizal (EM) communities from NH4+ and NO3- . We hypothesized that a proportional fraction of ectomycorrhizal N uptake is transferred to the host, thereby resulting in the same uptake patterns of plants and their associated mycorrhizal communities. 15 N uptake was studied under various field conditions after short-term and long-term exposure to a pulse of equimolar NH4+ and NO3- concentrations, where one compound was replaced by 15 N. In native EM assemblages, long-term and short-term 15 N uptake from NH4+ was higher than that from NO3- , regardless of season, water availability and site exposure, whereas in beech long-term 15 N uptake from NO3- was higher than that from NH4+ . The transfer rates from the EM to beech were lower for 15 N from NH4+ than from NO3- . 15 N content in EM was correlated with 15 N uptake of the host for 15 NH4+ , but not for 15 NO3- -derived N. These findings suggest stronger control of the EM assemblage on N provision to the host from NH4+ than from NO3- . Different host and EM accumulation patterns for inorganic N will result in complementary resource use, which might be advantageous in forest ecosystems with limited N availability.

Phosphorus is one of the major limiting factors of primary productivity in terrestrial ecosystems and, thus, the P demand of plants might be among the most important drivers of soil and ecosystem development. The P cycling in forest ecosystems seems an ideal example to illustrate the concept of ecosystem nutrition. Ecosystem nutrition combines and extents the traditional concepts of nutrient cycling and ecosystem ecology. The major extension is to consider also the loading and unloading of nutrient cycles and the impact of nutrient acquiring and recycling processes on overall ecosystem properties. Ecosystem nutrition aims to integrate nutrient related aspects at different scales and in different ecosystem compartments including all processes, interactions and feedbacks associated with the nutrition of an ecosystem. We review numerous previous studies dealing with P nutrition from this ecosystem nutrition perspective. The available information contributes to the description of basic ecosystem characteristics such as emergence, hierarchy, and robustness. In result, we were able to refine Odum's hypothesis on P nutrition strategies along ecosystem succession to substrate related ecosystem nutrition and development. We hypothesize that at sites rich in mineral-bound P, plant and microbial communities tend to introduce P from primary minerals into the biogeochemical P cycle (acquiring systems), and hence the tightness of the P cycle is of minor relevance for ecosystem functioning. In contrast, tight P recycling is a crucial emergent property of forest ecosystems established at sites poor in mineral bound P (recycling systems). We conclude that the integration of knowledge on nutrient cycling, soil science, and ecosystem ecology into holistic ecosystem nutrition will provide an entirely new view on soil–plant–microbe interactions.

This study aimed to investigate the shifts in net nitrogen (N) uptake and N compounds of fine roots over the vegetation period (i.e., spring, summer, autumn) and correlate this with NO concentration in the soil. Soil NO concentration was measured using gas lysimeters for collection and a chemiluminescence analyzer for quantification. Net N uptake by the roots was determined using the 15N enrichment technique. N pools were quantified using spectrophotometric techniques. Soil NO concentrations at beech and spruce forest sites were highest in spring (June), and lowest in winter (December). Total N of the roots was similar during the seasons and between the two years under study despite considerable variation of different N compounds. Net N uptake generally increased with higher N supply. Correlation analysis revealed a positive relationship between soil NO concentration and net N uptake only for spruce trees. This relationship seemed to be modulated by environmental factors and tree species.

Carnivorous Dionaea muscipula operates active snap traps for nutrient acquisition from prey; so what is the role of D. muscipula's reduced root system? We studied the capacity for nitrogen (N) acquisition via traps, and its effect on plant allometry; the capacity of roots to absorb NO3 (-) , NH4 (+) and glutamine from the soil solution; and the fate and interaction of foliar- and root-acquired N. Feeding D. muscipula snap traps with insects had little effect on the root:shoot ratio, but promoted petiole relative to trap growth. Large amounts of NH4 (+) and glutamine were absorbed upon root feeding. The high capacity for root N uptake was maintained upon feeding traps with glutamine. High root acquisition of NH4 (+) was mediated by 2.5-fold higher expression of the NH4 (+) transporter DmAMT1 in the roots compared with the traps. Electrophysiological studies confirmed a high constitutive capacity for NH4 (+) uptake by roots. Glutamine feeding of traps inhibited the influx of (15) N from root-absorbed (15) N/(13) C-glutamine into these traps, but not that of (13) C. Apparently, fed traps turned into carbon sinks that even acquired organic carbon from roots. N acquisition at the whole-plant level is fundamentally different in D. muscipula compared with noncarnivorous species, where foliar N influx down-regulates N uptake by roots.

Overexpression of bacterial γ-glutamylcysteine synthetase in the cytosol of Populus tremula × P. alba produces higher glutathione (GSH) concentrations in leaves, thereby indicating the potential for cadmium (Cd) phytoremediation. However, the net Cd(2+) influx in association with H(+) /Ca(2+) , Cd tolerance, and the underlying molecular and physiological mechanisms are uncharacterized in these poplars. We assessed net Cd(2+) influx, Cd tolerance and the transcriptional regulation of several genes involved in Cd(2+) transport and detoxification in wild-type and transgenic poplars. Poplars exhibited highest net Cd(2+) influxes into roots at pH 5.5 and 0.1 mM Ca(2+) . Transgenics had higher Cd(2+) uptake rates and elevated transcript levels of several genes involved in Cd(2+) transport and detoxification compared with wild-type poplars. Transgenics exhibited greater Cd accumulation in the aerial parts than wild-type plants in response to Cd(2+) exposure. Moreover, transgenic poplars had lower concentrations of O2 ˙(-) and H2 O2 ; higher concentrations of total thiols, GSH and oxidized GSH in roots and/or leaves; and stimulated foliar GSH reductase activity compared with wild-type plants. These results indicate that transgenics are more tolerant of 100 μM Cd(2+) than wild-type plants, probably due to the GSH-mediated induction of the transcription of genes involved in Cd(2+) transport and detoxification.

Sulphate uptake and its distribution within plants depend on the activity of different sulphate transporters (SULTR). In long-living deciduous plants such as trees, seasonal changes of spatial patterns add another layer of complexity to the question of how the interplay of different transporters adjusts S distribution within the plant to environmental changes. Poplar is an excellent model to address this question because its S metabolism is already well characterized. In the present study, the importance of SULTRs for seasonal sulphate storage and mobilization was examined in the wood of poplar (Populus tremula × P. alba) by analysing their gene expression in relation to sulphate contents in wood and xylem sap. According to these results, possible functions of the respective SULTRs for seasonal sulphate storage and mobilization in the wood are suggested. Together, the present results complement the previously published model for seasonal sulphate circulation between leaves and bark and provide information for future mechanistic modelling of whole tree sulphate fluxes.

We investigated the interaction of rhizospheric nitric oxide (NO) concentration (i.e. low, ambient or high) and soil nitrogen (N) availability (i.e. low or high) with organic and inorganic N uptake by fine roots of Pinus sylvestris L. seedlings by (15) N feeding experiments under controlled conditions. N metabolites in fine roots were analysed to link N uptake to N nutrition. NO affected N uptake depending on N source and soil N availability. The suppression of nitrate uptake in the presence of ammonium and glutamine was overruled by high NO. The effects of NO on N uptake with increasing N availability showed different patterns: (1) increasing N uptake regardless of NO concentration (i.e. ammonium); (2) increasing N uptake only with high NO concentration (i.e. nitrate and arginine); and (3) decreasing N uptake (i.e. glutamine). At low N availability and high NO nitrate accumulated in the roots indicating insufficient substrates for nitrate reduction or its storage in root vacuoles. Individual amino acid concentrations were negatively affected with increasing NO (i.e. asparagine and glutamine with low N availability, serine and proline with high N availability). In conclusion, this study provides first evidence that NO affects N uptake and metabolism in a conifer.

Climate-tolerant tree species and/or provenances have to be selected to ensure the high productivity of managed forests in Central Europe under the prognosticated climate changes. For this purpose, we studied the responses of saplings from three oak species (i.e. Quercus robur, Q.�petraea and Q.�pubescens) and provenances of different climatic origin (i.e. low or high rainfall, low or high temperature habitats) with regard to leaf nitrogen (N) composition as a measure of N nutrition. Saplings were grown in model ecosystems on either calcareous or acidic soil and subjected to one of four treatments (control, drought, air warming or a combination of drought and air warming). Across species, oak N metabolism responded to the influence of drought and/or air warming with an increase in leaf amino acid N concentration at the expense of structural N. Moreover, provenances or species from drier habitats were more tolerant to the climate conditions applied, as indicated by an increase in amino acid N (comparing species) or soluble protein N (comparing provenances within a species). Furthermore, amino acid N concentrations of oak leaves were significantly higher on calcareous compared to acidic soil. From these results, it can be concluded that seeds from provenances or species originating from drier habitats and ? if available ? from calcareous soil types may provide a superior seed source for future forest establishment.

This study aimed to identify drought-mediated differences in amino nitrogen (N) composition and content of xylem and phloem in trees having different symbiotic N(2)-fixing bacteria. Under controlled water availability, 1-year-old seedlings of Robinia pseudoacacia (nodules with Rhizobium), Hippophae rhamnoides (symbiosis with Frankia) and Buddleja alternifolia (no such root symbiosis) were exposed to control, medium drought and severe drought, corresponding soil water content of 70-75%, 45-50% and 30-35% of field capacity, respectively. Composition and content of amino compounds in xylem sap and phloem exudates were analysed as a measure of N nutrition. Drought strongly reduced biomass accumulation in all species, but amino N content in xylem and phloem remained unaffected only in R. pseudoacacia. In H. rhamnoides and B. alternifolia, amino N in phloem remained constant, but increased in xylem of both species in response to drought. There were differences in composition of amino compounds in xylem and phloem of the three species in response to drought. Proline concentrations in long-distance transport pathways of all three species were very low, below the limit of detection in phloem of H. rhamnoides and in phloem and xylem of B. alternifolia. Apparently, drought-mediated changes in N composition were much more connected with species-specific changes in C:N ratios. Irrespective of soil water content, the two species with root symbioses did not show similar features for the different types of symbiosis, neither in N composition nor in N content. There was no immediate correlation between symbiotic N fixation and drought-mediated changes in amino N in the transport pathways.

• Increasing atmospheric concentrations of phytotoxic ozone (O(3) ) can constrain growth and carbon sink strength of forest trees, potentially exacerbating global radiative forcing. Despite progress in the conceptual understanding of the impact of O(3) on plants, it is still difficult to detect response patterns at the leaf level. • Here, we employed principal component analysis (PCA) to analyse a database containing physiological leaf-level parameters of 60-yr-old Fagus sylvatica (European beech) trees. Data were collected over two climatically contrasting years under ambient and twice-ambient O(3) regimes in a free-air forest environment. • The first principal component (PC1) of the PCA was consistently responsive to O(3) and crown position within the trees over both years. Only a few of the original parameters showed an O(3) effect. PC1 was related to parameters indicative of oxidative stress signalling and changes in carbohydrate metabolism. PC1 correlated with cumulative O(3) uptake over preceding days. • PC1 represents an O(3) -responsive multivariate pattern detectable in the absence of consistently measurable O(3) effects on individual leaf-level parameters. An underlying effect of O(3) on physiological processes is indicated, providing experimental confirmation of theoretical O(3) response patterns suggested previously.

In the present study, the significance of sulfite oxidase (SO) for sulfite detoxification and sulfur assimilation was investigated. In response to sulfur dioxide (SO(2)) exposure, a remarkable expansion of sulfate and a significant increase of GSH pool were observed in wild-type and SO-overexpressing Arabidopsis. These metabolic changes were connected with a negative feedback inhibition of adenosine 5'-phosphosulfate reductase (APR), but no alterations in gas exchange parameters or visible symptoms of injury. However, Arabidopsis SO-KO mutants were consistently negatively affected upon 600 nL L(-1) SO(2) exposure for 60 h and showed phenotypical symptoms of injury with small necrotic spots on the leaves. The mean g(H2O) was reduced by about 60% over the fumigation period, accompanied by a reduction of net CO(2) assimilation and SO(2) uptake of about 50 and 35%. Moreover, sulfur metabolism was completely distorted. Whereas sulfate pool was kept constant, thiol-levels strongly increased. This demonstrates that SO should be the only protagonist for back-oxidizing and detoxification of sulfite. Based on these results, it is suggested that co-regulation of SO and APR controls sulfate assimilation pathway and stabilizes sulfite distribution into organic sulfur compounds. In conclusion, a sulfate-sulfite cycle driven by APR and SO can be postulated for fine-tuning of sulfur distribution that is additionally used for sulfite detoxification, when plants are exposed to atmospheric SO(2).

Recent studies of transgenic poplars over-expressing the genes gsh1 and gsh2 encoding γ-glutamylcysteine synthetase (γ-ECS) and glutathione synthetase, respectively, provided detailed information on regulation of GSH synthesis, enzymes activities and mRNA expression. In this experiment, we studied quantitative parameters of leaves, assimilating tissues, cells and chloroplasts, mesophyll resistance for CO(2) diffusion, chlorophyll and carbohydrate content in wild-type poplar and transgenic plants over-expressing gsh1 in the cytosol after 3 years of growth in relatively clean (control) or heavy metal-contaminated soil in the field. Over-expression of gsh1 in the cytosol led to a twofold increase of intrafoliar GSH concentration and influenced the photosynthetic apparatus at different levels of organisation, i.e., leaves, photosynthetic cells and chloroplasts. At the control site, transgenic poplars had a twofold smaller total leaf area per plant and a 1.6-fold leaf area per leaf compared to wild-type controls. Annual aboveground biomass gain was reduced by 50% in the transgenic plants. The reduction of leaf area of the transformants was accompanied by a significant decline in total cell number per leaf, indicating suppression of cell division. Over-expression of γ-ECS in the cytosol also caused changes in mesophyll structure, i.e., a 20% decrease in cell and chloroplast number per leaf area, but also an enhanced volume share of chloroplasts and intercellular airspaces in the leaves. Transgenic and wild poplars did not exhibit differences in chlorophyll and carotenoid content of leaves, but transformants had 1.3-fold fewer soluble carbohydrates. Cultivation on contaminated soil caused a reduction of palisade cell volume and chloroplast number, both per cell and leaf area, in wild-type plants but not in transformants. Biomass accumulation of wild-type poplars decreased in contaminated soil by more than 30-fold, whereas transformants showed a twofold decrease compared to the control site. Thus, poplars over-expressing γ-ECS in the cytosol were more tolerant to heavy metal stress under field conditions than wild-type plants according to the parameters analysed. Correlation analysis revealed strong dependence of cell number per leaf area unit, chloroplast parameters and mesophyll resistance with the GSH level in poplar leaves.

Mangrove sediments can act as sources of the greenhouse trace gases, nitrous oxide (N2O) and methane (CH4). Confident reporting of trace gas emissions from mangrove sediments at local levels is important for regional emissions inventories, since small changes in N2O and CH4 fluxes greatly influence greenhouse gas budgets due to their high global warming potentials. It is also important to identify the drivers of trace gas emission, to prioritise management for minimising emissions. We measured N2O and CH4 fluxes and abiotic sediment parameters at midday low tide in winter and summer seasons, at four sites (27 degrees 33'S, 152 degrees 59'E) ranging from estuary to ocean sub-tropical mangrove sediments, having varied anthropogenic impacts. At all sites, sediment N2O and CH4 emissions were significantly lower during winter (7-26 mu g N2O m-2 center dot h-1; 47-466 mu g CH4 m-2 center dot h-1) compared to summer (28-202 mu g N2O m-2 center dot h-1; 247-1570 mu g CH4 m-2 center dot h-1). Sediment temperature, ranging from 18 to 33 degrees C, strongly influenced N2O and CH4 emissions. Highest emissions (202 mu g N2O m-2 center dot h-1, 1570 mu g CH4 m-2 center dot h-1) were detected at human-impacted estuary sites, which generally had higher total carbon (< 8%) and total nitrogen (< 0.4%) in sediments and reduced salinity (< 16 dS center dot m-1). Large between-site variation highlights the need for regular monitoring of sub-tropical mangroves to capture short-lived, episodic N2O and CH4 flux events that are affected by sediment biophysico-chemical conditions at site level. This is important, particularly at sites receiving anthropogenic nutrients, and that have variable freshwater inputs and tidal hydrology.

To investigate the short-term consequences of direct competition between beech and sycamore maple on root N uptake and N composition, mycorrhizal seedlings of both tree species were incubated for 4 days (i.e. beech only, sycamore maple only or both together) in an artificial nutrient solution with low N availability. On the fourth day, N uptake experiments were conducted to study the effects of competition on inorganic and organic N uptake. For this purpose, multiple N sources were applied with a single label. Furthermore, fine roots were sampled and analysed for total amino acids, soluble protein, total nitrogen, nitrate and ammonium content. Our results clearly show that both tree species were able to use inorganic and organic N sources. Uptake of inorganic and organic N by beech roots was negatively affected in the presence of the competing tree species. In contrast, the presence of beech stimulated inorganic N uptake by sycamore maple roots. Both the negative effect of sycamore maple on N uptake of beech and the positive effect of beech on N uptake of sycamore maple led to an increase in root soluble protein in beech, despite an overall decrease in total N concentration. Thus, beech compensated for the negative effects of the tree competitor on N uptake by incorporating less N into structural N components, but otherwise exhibited the same strategy as the competitor, namely, enhancing soluble protein levels in roots when grown under competition. It is speculated that enhanced enzyme activities of so far unknown nature are required in beech as a defence response to inter-specific competition.

Many forest ecosystems have evolved at sites with growth-limiting nitrogen (N) availability, low N input from external sources and high ecosystem internal cycling of N. By contrast, many poplar species are frequent constituents of floodplain forests where they are exposed to a significant ecosystem external supply of N, mainly nitrate, in the moving water table. Therefore, nitrate is much more important for N nutrition of these poplar species than for many other tree species. We summarise current knowledge of nitrate uptake and its regulation by tree internal signals, as well as acquisition of ammonium and organic N from the soil. Unlike herbaceous plants, N nutrition of trees is sustained by seasonal, tree internal cycling. Recent advances in the understanding of seasonal storage and mobilisation in poplar bark and regulation of these processes by temperature and daylength are addressed. To explore consequences of global climate change on N nutrition of poplar trees, responses of N uptake and metabolism to increased atmospheric CO(2) and O(3) concentrations, increased air and soil temperatures, drought and salt stress are highlighted.

Forest ecosystems with low soil nitrogen (N) availability are characterized by direct competition for this growth-limiting resource between several players, i.e. various components of vegetation, such as old-growth trees, natural regeneration and understorey species, mycorrhizal fungi, free-living fungi and bacteria. With the increase in frequency and intensity of extreme climate events predicted in current climate change scenarios, also competition for N between plants and/or soil microorganisms will be affected. In this review, we summarize the present understanding of ecosystem N cycling in N-limited forests and its interaction with extreme climate events, such as heat, drought and flooding. More specifically, the impacts of environmental stresses on microbial release and consumption of bioavailable N, N uptake and competition between plants, as well as plant and microbial uptake are presented. Furthermore, the consequences of drying-wetting cycles on N cycling are discussed. Additionally, we highlight the current methodological difficulties that limit present understanding of N cycling in forest ecosystems and the need for interdisciplinary studies.

Long-term nitrogen deposition into forest ecosystems has turned many forests in Central Europe and North America from N-limited to N-saturated systems, with consequences for climate as well as air and groundwater quality. However, complete quantification of processes that convert the N deposited and contributed to ecosystem N cycling is scarce. In this study, we provide the first complete quantification of external and internal N fluxes in an old-growth spruce forest, the Höglwald, Bavaria, Germany, exposed to high chronic N deposition. In this forest, N cycling is dominated by high rates of mineralisation of soil organic matter, nitrification and immobilisation of ammonium and nitrate into microbial biomass. The amount of ammonium available is sufficient to cover the entire N demand of the spruce trees. The data demonstrate the existence of a highly dynamic internal N cycle within the soil, driven by growth and death of the microbial biomass, which turns over approximately seven times each year. Although input and output fluxes are of high environmental significance, they are low compared to the internal fluxes mediated by microbial activity.

Poplar mutants overexpressing the bacterial genes gsh1 or gsh2 encoding the enzymes of glutathione biosynthesis are among the best-characterised transgenic plants. However, this characterisation originates exclusively from laboratory studies, and the performance of these mutants under field conditions is largely unknown. Here, we report a field experiment in which the wild-type poplar hybrid Populus tremula x P. alba and a transgenic line overexpressing the bacterial gene gsh1 encoding gamma-glutamylcysteine synthetase in the cytosol were grown for 3 years at a relatively clean (control) field site and a field site contaminated with heavy metals. Aboveground biomass accumulation was slightly smaller in transgenic compared to wild-type plants; soil contamination significantly decreased biomass accumulation in both wild-type and transgenic plants by more than 40%. Chloroplasts parameters, i.e., maximal diameter, projection area and perimeter, surface area and volume, surface/volume ratio and a two-dimensional form coefficient, were found to depend on plant type, leaf tissue and soil contamination. The greatest differences between wild and transgenic poplars were observed at the control site. Under these conditions, chloroplast sizes in palisade tissue of transgenic poplar significantly exceeded those of the wild type. In contrast to the wild type, palisade chloroplast volume exceeded that of spongy chloroplasts in transgenic poplars at both field sites. Chlorophyll content per chloroplast was the same in wild and transgenic poplars. Apparently, the increase in chloroplast volume was not connected to changes in the photosynthetic centres. Chloroplasts of transgenic poplar at the control site were more elongated in palisade cells and close to spherical in spongy mesophyll chloroplasts. At the contaminated site, palisade and spongy cell chloroplasts of leaves from transgenic trees and the wild type were the same shape. Transgenic poplars also had a smaller chloroplast surface/volume ratio, both at the control and the contaminated site. Chloroplast number per cell did not differ between wild and transgenic poplars at the control site. Soil contamination led to suppression of chloroplast replication in wild-type plants. From these results, we assume that overexpressing the bacterial gsh1 gene in the cytosol interacts with processes in the chloroplast and that sequestration of heavy metal phytochelatin complexes into the vacuole may partially counteract this interaction in plants grown at heavy metal-contaminated field sites. Further experiments are required to test these assumptions.

Tree and shrub species can be differentiated into two major groups based on their substantially different leaf anatomy: heterobaric and homobaric. In contrast to homobaric leaves, heterobaric leaves have bundle sheath extensions (BSEs) that create transparent regions on their lamina. Recent studies have shown that BSEs transfer visible light to internal mesophyll layers, thus affecting the photosynthetic performance of heterobaric leaves. Whether the two leaf types also differ in other functional and structural traits has not been addressed, nor have any structure – function relations. Here, we measured key anatomical and physiological parameters and tested their relationships in 30 species with different leaf types. Heterobaric leaves were thinner with lower leaf mass per area, had higher nitrogen concentration per mass, were 13 C-enriched, and achieved comparable photosynthetic capacity per area but had higher photosynthetic capacity per mass compared to homobaric leaves. Relations between leaf construction cost, nitrogen concentration, and photosynthesis followed the general pattern of the “ leaf economic spectrum, ” but differed between homobaric and heterobaric leaves. We suggest that the mechanisms controlling these relations differ between the two leaf types, presumably due to their distinct anatomy.

Nitrogen nutrition and salt stress experiments were performed in a greenhouse with hydroponic-cultured, salt-sensitive Grey poplar (Populus x canescens) plants to study the combined influence of different N sources (either 1 mm NO(3) (-) or NH(4)(+)) and salt (up to 75 mm NaCl) on leaf gas exchange, isoprene biosynthesis and VOC emissions. Net assimilation and transpiration proved to be highly sensitive to salt stress and were reduced by approximately 90% at leaf sodium concentrations higher than 1,800 microg Na g dry weight (dw)(-1). In contrast, emissions of isoprene and oxygenated VOC (i.e. acetaldehyde, formaldehyde and acetone) were unaffected. There was no significant effect of combinations of salt stress and N source, and neither NO(3)(-) or NH(4)(+) influenced the salt stress response in the Grey poplar leaves. Also, transcript levels of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (PcDXR) and isoprene synthase (PcISPS) did not respond to the different N sources and only responded slightly to salt application, although isoprene synthase (PcISPS) activity was negatively affected at least in one of two experiments, despite high isoprene emission rates. A significant salt effect was the strong reduction of leaf dimethylallyl diphosphate (DMADP) content, probably due to restricted availability of photosynthates for DMADP biosynthesis. Further consequences of reduced photosynthetic gas exchange and maintaining VOC emissions are a very high C loss, up to 50%, from VOC emissions related to net CO(2) uptake and a strong increase in leaf internal isoprene concentrations, with maximum mean values up to 6.6 microl x l(-1). Why poplar leaves maintain VOC biosynthesis and emission under salt stress conditions, despite impaired photosynthetic CO(2) fixation, is discussed.

The significance of root nitrate reductase for sulfur assimilation was studied in tobacco (NICOTIANA TABACUM) plants. For this purpose, uptake, assimilation, and long-distance transport of sulfur were compared between wild-type tobacco and transformants lacking root nitrate reductase, cultivated either with nitrate or with ammonium nitrate. A recently developed empirical model of plant internal nitrogen cycling was adapted to sulfur and applied to characterise whole plant sulfur relations in wild-type tobacco and the transformant. Both transformation and nitrogen nutrition strongly affected sulfur pools and sulfur fluxes. Transformation decreased the rate of sulfate uptake in nitrate-grown plants and root sulfate and total sulfur contents in root biomass, irrespective of N nutrition. Nevertheless, glutathione levels were enhanced in the roots of transformed plants. This may be a consequence of enhanced APR activity in the leaves that also resulted in enhanced organic sulfur content in the leaves of the tranformants. The lack of nitrate reductase in the roots in the transformants caused regulatory changes in sulfur metabolism that resembled those observed under nitrogen deficiency. Nitrate nutrition reduced total sulfur content and all the major fractions analysed in the leaves, but not in the roots, compared to ammonium nitrate supply. The enhanced organic sulfur and glutathione levels in ammonium nitrate-fed plants corresponded well to elevated APR activity. But foliar sulfate contents also increased due to decreased re-allocation of sulfate into the phloem of ammonium nitrate-fed plants. Further studies will elucidate whether this decrease is achieved by downregulation of a specific sulfate transporter in vascular tissues.

Databases are needed for the ozone (O 3 ) risk assessment on adult forest trees under stand conditions, as mostly juvenile trees have been studied in chamber experiments. A synopsis is presented here from an integrated case study which was conducted on adult FAGUS SYLVATICA trees at a Central-European forest site. Employed was a novel free-air canopy O 3 fumigation methodology which ensured a whole-plant assessment of O 3 sensitivity of the about 30 m tall and 60 years old trees, comparing responses to an experimental 2 × ambient O 3 regime (2 × O 3 , max. 150 nl O 3 l -1 ) with those to the unchanged 1 × ambient O 3 regime (1 × O 3 = control) prevailing at the site. Additional experimentation on individual branches and juvenile beech trees exposed within the forest canopy allowed for evaluating the representativeness of young-tree and branch-bag approaches relative to the O 3 sensitivity of the adult trees. The 2 × O 3 regime did not substantially weaken the carbon sink strength of the adult beech trees, given the absence of a statistically significant decline in annual stem growth; a 3 % reduction across five years was demonstrated, however, through modelling upon parameterization with the elaborated database. 2 × O 3 did induce a number of statistically significant tree responses at the cell and leaf level, although the O 3 responsiveness varied between years. Shade leaves displayed an O 3 sensitivity similar to that of sun leaves, while indirect belowground O 3 effects, apparently mediated through hormonal relationships, were reflected by stimulated fine-root and ectomycorrhizal development. Juvenile trees were not reliable surrogates of adult ones in view of O 3 risk assessment. Branch sections enclosed in (climatized) cuvettes, however, turned out to represent the O 3 sensitivity of entire tree crowns. Drought-induced stomatal closure decoupled O 3 intake from O 3 exposure, as in addition, also the “physiologically effective O 3 dose” was subject to change. No evidence emerged for a need to lower the “Critical Level for Ozone” in risk assessment of forest trees, although sensitive tree parameters did not necessarily reflect a linear relationship to O 3 stress. Exposure-based concepts tended to overestimate O 3 risk under drought, which is in support of current efforts to establish flux-related concepts of O 3 intake in risk assessment.

The influence of long-term free-air ozone fumigation and canopy position on leaf contents of total glutathione, its redox state, non-structural proteins (NSP), soluble amino compounds, and total soluble sugars in old-growth beech (FAGUS SYLVATICA) and spruce (PICEA ABIES) trees were determined over a period of five years. Ozone fumigation had weak effects on the analysed metabolites of both tree species and significant changes in the contents of total glutathione, NSP, and soluble sugars were observed only selectively. Beech leaves were affected by crown position to a higher extent than spruce needles and exhibited lower contents of total glutathione and NSP and total soluble sugars, but enhanced contents of oxidised glutathione and amino compounds in the shade compared to the sun crown. Contents of total soluble sugars generally were decreased in shade compared to sun needles of spruce trees. Interspecific differences between beech and spruce were more distinct in the sun compared to the shade crown. Contents of total glutathione were increased whilst contents of amino compounds and total soluble sugars were lower in spruce needles compared to beech leaves. The metabolites determined showed individual patterns in the course of the five measurement years. Contents of total glutathione and its redox state correlated with air temperature and global radiation, indicating an important role for the antioxidant at low temperatures. Correlations of glutathione with instantaneous ozone concentrations seem to be a secondary effect. Differences in proteins and/or amino compounds in the inter-annual course are assumed to be a consequence of alterations in specific N uptake rates.

To test the effects of calcium on wood formation, Populus tremula x Populus tremuloides clones were supplied with Hoagland solution modified in its calcium contents. Energy-dispersive X-ray analysis (EDXA) revealed an increase in calcium in the phloem, the cambium and the xylem elongation zone with increasing Ca(2+) supply in the nutrient solution. Using light and electron microscopy, a strong impact was shown on the cambial and the elongation zones under calcium starvation. Using Fourier transform infrared (FTIR) spectroscopy on wood and bark cells formed under calcium starvation, we detected a reduction of some absorptions, such as carbonyl and methoxy groups from S-lignin. Also, a significant reduction in fiber length was detected with decreasing calcium supply in the nutrient solution. High-performance liquid chromatography (HPLC) analysis revealed a large increase in sugar concentrations in the leaves, but reduced concentrations in the bark under Ca(2+) deficiency. In conclusion, our results show a significant influence of calcium on the structure, chemistry and physiology of wood formation. Thus, efficient Ca(2+) supply has to be considered a decisive factor in wood formation.

This study investigated the influence of meteorological, pedospheric and physiological factors on the water relations of Scots pine, as characterized by the origin of water taken up, by xylem transport as well as by carbon isotope discrimination (Delta13C) and oxygen isotope enrichment (Delta18O) of newly assimilated organic matter. For more than 1 year, we quantified delta2H and delta18O of potential water sources and xylem water as well as Delta13C and Delta18O in twig and trunk phloem organic matter biweekly, and related these values to continuously measured or modelled meteorological parameters, soil water content, stand transpiration (ST) and canopy stomatal conductance (G(s)). During the growing season, delta18O and delta2H of xylem water were generally in a range comparable to soil water from a depth of 2-20 cm. Long residence time of water in the tracheids uncoupled the isotopic signals of xylem and soil water in winter. Delta18O but not Delta13C in phloem organic matter was directly indicative of recent environmental conditions during the whole year. Delta18O could be described applying a model that included 18O fractionation associated with water exchange between leaf and atmosphere, and with the production of organic matter as well as the influence of transpiration. Phloem Delta13C was assumed to be concertedly influenced by G(s) and photosynthetically active radiation (PAR) (as a proxy for photosynthetic capacity). We conclude that isotope signatures can be used as effective tools (1) to characterize the seasonal dynamics in source and xylem water, and (2) to assess environmental effects on transpiration and G(s) of Scots pine, thus helping to understand and predict potential impacts of climate change on trees and forest ecosystems.

We aimed to quantify the separate effects of photosynthetic and postphotosynthetic carbon isotope discrimination on d 13 C of the fast-turn-over carbon pool (water soluble organic carbon and CO2 emitted from heterotrophic tissues), including their diel variation, along the pathway of carbon transport from the foliage to the base of the stem. For that purpose, we determined d 13 C in total and water-soluble organic matter of the foliage plus d 13 C and d 18 O in phloem organic matter of twigs and at three heights along the stem of Pinus sylvestris over a nine-day period, including four measurements per day. These data were related to meteorological and photosynthesis parameters and to the d 13 C of stem-emitted CO2. In the canopy (foliage and twigs), the d 13 C of soluble organic matter varied diurnally with amplitudes of up to 1.9%. The greatest 13 C enrichment was recorded during the night/early morning, indicating a strong influence of starch storage and remobilization on the carbon isotope signatures of sugars exported from the leaves. 13 C enrichment of soluble organic matter from the leaves to the twig phloem and further on to the phloem of the stem was supposed to be a result of carbon isotope fractionation associated with metabolic processes in the source and sink tissues. CO2 emitted from the stem was enriched by 2.3‐5.2% compared with phloem organic matter. When day-to-day variation was addressed, water-soluble leaf d 13 C and twig phloem d 18 O were strongly influenced by ci/ca and stomatal conductance (Gs), respectively. These results show that both photosynthetic and postphotosynthetic carbon isotope fractionation influence d 13 C of organic matter over time, and over the length of the basipetal transport pathway. Clearly, these influences on the d 13 C of respired CO2 must be considered when using the latter for partitioning of ecosystem CO2 fluxes or when the assessment of d 13 C in organic matter is applied to estimate environmental effects in ci/ca.

Beech seedlings from 11 German climatic provenances were exposed to a realistically timed drought treatment in a greenhouse experiment. The stable isotope composition of carbon (C) and nitrogen (N) was analysed in pooled bulk material of roots, stems and leaves, as well as in the aqueous extracts and starch fractions. The delta 13C values increased in bulk samples (BS) of roots, stems and leaves by drought, although no leaf growth occurred during the experimental period. A clear drought effect on delta 13C in aqueous extracts was detected in leaves. In aqueous extracts of stems and roots as well as in starch fractions of all organs, abundance of delta 13C also tended to be increased by drought, but this effect was not statistically significant. For both delta 13C and delta 15N, enrichment was observed from the site of uptake/ source to the site of use/sink. A gradient for delta 13C in all fractions from leaves (-29.49, -28.89 and -27.85 per thousand) to stems (-28.81, -27.48 and -26.98 per thousand) and to roots (-27.60, -26.37 and -26.48 per thousand) was detected in BS, aqueous extracts and starch, respectively. An opposite gradient for delta 15N was found in BS: 1.59 per thousand, 1.84 per thousand and 3.05 per thousand in roots, stems and leaves, respectively. delta 15N was neither affected by drought in the BS nor in aqueous extracts, but an effect of provenance was observed. Particularly in roots and stems, drought-sensitive provenances showed the strongest shifts in delta 13C induced by drought and the lowest delta 15N values. In the present experiment, delta 13C values were more affected by the environmental factor drought, while delta 15N values were more affected by the genetic factor provenance.

To assess the physiological performance of drought-sensitive European beech ( Fagus sylvatica L.) under the dry Mediterranean climate prevailing at its southeastern distribution limit in Europe, we analyzed seasonal changes in carbon, nitrogen and water balance of naturally grown adult trees. We determined the foliar C and N contents, delta13C and delta18O signatures, total soluble non-protein nitrogen compounds (TSNN) in xylem, leaves, and phloem, as well as leaf water potential and photosynthetic quantum yield in northern Greece during 2003. Tissue sampling was performed in May, July, and September, while field measurements were conducted regularly. Climatic conditions for the 2003 growing season fall within the typical range of the studied area. The N- and C-related parameters displayed distinct seasonal courses. TSNN was highest in May in all tissues, and asparagine (Asn) was then the most abundant compound. Thereafter, TSNN decreased significantly in all tissues and both its concentration and composition remained constant in July and September. In both months, glutamate (Glu) prevailed in leaves, gamma-aminobutyric acid (GABA) in phloem exudates from twigs and trunks, and arginine (Arg) in the xylem sap, where loading with amino acids was rather low during that period, amounting to only 0.8 micromol N ml-1 in September. Highest total foliar N and C contents were detected in May, and the elevated abundance of nutrients as well as an increased foliar delta13C signature at the beginning of the growing season is attributed to remobilization processes. The signatures of delta18O, quantum yield and leaf water potentials varied only slightly throughout the growing season. Although summer precipitation at the study site was considerably lower compared to what is usual for typical central European beech forests, no intensive drought responses of the physiological apparatus were detected in the studied beech trees. This suggests efficient internal regulation mechanisms, constantly ensuring a favourable physiological status under the relatively dry Mediterranean climate.

Under the site conditions of a temperate forest, the exchange of short-chained oxygenated carbonyls (aldehydes, ketones) was assessed from leaves of adult European beech trees. The crowns of the trees were either exposed to an elevated O 3 regime as released by a free-air fumigation system (2 x O 3 ) or to the unchanged O 3 regime at the site (1 x O 3 , 'control'). Daily fluctuations of oxygenated carbonyls were quantified in relation to environmental and physiological factors. In particular, the effect of O 3 on carbonyl exchange was studied. Measurements of leaf gas exchange were performed with a dynamic cuvette system, and carbonyl fluxes were determined using 2,4-dinitrophenylhydrazine (DNPH)-coated silica gel cartridges. Leaves mainly emitted acetaldehyde, formaldehyde and acetone. Acetaldehyde dominated the emissions, amounting up to 100 nmol m -2 min -1 , followed by formaldehyde (approximately 80 nmol m -2 min -1 ) and acetone (approximately 60 nmol m -2 min -1 ). Carbonyl emissions were highest during midday and significantly lowered at night, irrespective of the O 3 exposure regime. Trees exposed to 2 x O 3 emitted acetaldehyde and acetone at enhanced rates. The findings are of particular significance for future climate change scenarios that assume increased O 3 levels.

The potential negative influence of competition from early successional species like blackberry (Rubus fruticosus) may be decisive for the natural regeneration success of drought-sensitive beech (Fagus sylvatica), especially in the light of climate change. With a split plot glasshouse experiment, we investigated the influence of two air temperature and irradiance levels on the competitive interference of blackberry on the water, nitrogen (N) and carbon (C) balance of beech seedlings under moderate drought. When increased temperature was accompanied by low irradiance the biomass, root-to-shoot ratio, N uptake and assimilation rates of blackberry were lower compared with beech, either grown alone or with blackberry. By contrast, when elevated temperature and high irradiance were combined, the root-to-shoot ratio and specific N uptake rate of blackberry were substantially increased, while the N acquisition of beech was impaired. Under lower temperature, with either full light or shade, the presence of blackberry had no significant effects on beech, for almost all tested parameters. Under elevated air temperature beech was impaired by the presence of blackberry at high irradiance. These findings emphasize the interacting effects between environmental factors and competition on the establishment of beech regeneration, which should be considered for future forest management in the frame of climate change.

The analysis of stable isotope composition (delta13C, delta15N, delta18O) of phloem-transported organic matter is a useful tool for assessing short-term carbon and water balance of trees. A major constraint of the general application of this method to trees at natural field sites is that the collection of phloem sap with the "phloem bleeding" technique is restricted to particular species and plant parts. To overcome this restriction, we compared the contents (amino compounds and sugars) and isotope signatures (delta13C, delta15N, delta18O) of phloem sap directly obtained from incisions in the bark (bleeding technique) with phloem exudates where bark pieces were incubated in aqueous solutions (phloem exudation technique with and without chelating agents [EDTA, polyphosphate] in the initial sampling solution, which prevent blocking of sieve tubes). A comparable spectrum of amino compounds and sugars was detected using the different techniques. O, C, or N compounds in the initial sampling solution originating from the chelating agents always decreased precision of determination of the respective isotopic signatures, as indicated by higher standard deviation, and/or led to a significant difference of mean delta as compared to the phloem bleeding technique. Hence, depending on the element from which the ratio of heavy to light isotope is determined, compounds lacking C, N, and/or O should be used as chelating agents in the exudation solution. In applying the different techniques, delta13C of organic compounds transported in the phloem of the twig (exudation technique with polyphosphate as chelating agent) were compared with those in the phloem of the main stem (phloem bleeding technique) in order to assess possible differences in carbon isotope composition of phloem carbohydrates along the tree axis. In July, organic compounds in the stem phloem were significantly enriched in 13C by > 1.3 per thousand as compared to the twig phloem, whereas this effect was not observed in September. Correlation analysis between delta13C and stomatal conductance (Gs) revealed the gradient from the twigs to the stem observed in July may be attributed to temporal differences rather than to spatial differences in carbon isotope composition of sugars. As various authors have produced conflicting results regarding the enrichment/depletion of 13C in organic compounds in the leaf-to-stem transition, the different techniques presented in this paper can be used to provide further insight into fractionation processes associated with transport of C compounds from leaves to branches and down the main stem.

In the present study, important components of carbon metabolism of mature leaves of young poplar trees (Populus x canescens) were determined. Carbohydrate concentrations in leaves and xylem sap were quantified at five different times during the day and compared with photosynthetic gas exchange measurements (net assimilation, transpiration and rates of isoprene emission). Continuously measured xylem sap flow rates, with a time resolution of 15 min, were used to calculate diurnal balances of carbon metabolism of whole mature poplar leaves on different days. Loss of photosynthetically fixed carbon by isoprene emission and dark respiration amounted to 1% and 20%. The most abundant soluble carbohydrates in leaves and xylem sap were glucose, fructose and sucrose, with amounts of approx. 2 to 12 mmol m(-2) leaf area in leaves and about 0.2 to 15 mM in xylem sap. Clear diurnal patterns of carbohydrate concentration in xylem sap and leaves, however, were not observed. Calculations of the carbon transport rates in the xylem to the leaves were based on carbohydrate concentrations in xylem sap and xylem sap flow rates. This carbon delivery amounted to about 3 micromol C m(-2) s(-1) during the day and approx. 1 micromol C m(-2) s(-1) at night. The data demonstrated that between 9 and 28 % of total carbon delivered to poplar leaves during 24 h resulted from xylem transport and, hence, provide a strong indication for a significant rate of carbon cycling within young trees.

In the present field study we analysed the seasonal pattern of carbohydrate composition and contents in the xylem sap of Viscum album and the xylem sap of a deciduous (Populus x euramericana) and a coniferous (Abies alba) host tree species. The results were compared with the soluble carbohydrate composition and contents of mistletoe tissues. On both hosts significant amounts of glucose, fructose, and sucrose were found in the xylem sap of Viscum throughout the seasons. The general seasonal pattern of sugar contents, i.e. high concentrations in spring and lower concentrations in other seasons on Populus, and intermediate concentrations throughout the year on Abies, largely reflected the xylem sap carbohydrate composition and contents of the respective host. These observations provide indirect evidence for carbohydrate flux from the xylem sap of the host into the mistletoe. However, in both hosts xylem sap seems to be deviated into the mistletoe without specific control of carbohydrate flux. Differences observed between the seasonal pattern of xylem sap carbohydrate concentrations in Viscum on Populus and Abies may originate from the different time of leaf development of these species. A clear-cut seasonal pattern of soluble carbohydrates was not observed in the leaves of Viscum on both hosts. Still soluble carbohydrates seem to be reallocated from the senescing to the newly developed leaves of Viscum indicating that the seasonal requirement of carbohydrate for growth and development can only completely be met by carbohydrate acquisition from the host and their own photosynthesis.

Diurnal changes in starch, sugar and amino acid concentrations in source leaves, sink leaves and roots of tobacco plants were determined. In addition to wild type tobacco, transformed plants deficient in root nitrate reductase and exhibiting decreased rates of growth were employed. Further, the growth rates of tobacco plants were modulated by exposure to elevated pCO2. From the diurnal alterations in metabolite concentrations, the daily turnover of starch and amino N was estimated in order to: (i) elucidate whether turnover rates can be related to growth rates, and (ii) identify individual amino compounds with the potential to indicate nitrogen fluxes and the C/N status of plants. Elevated pCO2 increased growth rates and daily turnover of starch in both wild type and transformed plants, indicating enhanced rates of photosynthesis. In wild type plants, elevated pCO2 increased the turnover of amino N, notably glutamine and alanine, in mature source leaves, indicating enhanced nitrate reduction. By contrast, amino N turnover in source leaves of transformed plants was not affected by elevated pCO2, although nitrate reduction was presumably enhanced. Apparently, export of amino N was increased from the source leaves of transformed plants. This assumption was supported by a significantly increased turnover of amino N in young sink leaves compared to mature source leaves, indicating a preference for acropetal amino N allocation and import into the young leaves of the transformed plants. Further, elevated pCO2 increased the allocation of leaf-derived amino N to the roots of transformed plants. This led to increased levels of amino compounds during the entire day, notably glutamate, but did not affect root growth of the transformed plants. The suitability of individual amino compounds as markers for major N fluxes, such as nitrate reduction, photorespiration, and amino N export and import is discussed.

Pedospheric nitrate uptake is a strongly regulated process adapted to the N demand of the whole plant. Pre-requisites for an integrating regulatory system are signal substances communicating the N demand of the shoot to the roots. In the current study it was shown that an additional atmospheric N source results in activation of cytokinins in the shoot as indicated by an increase in the trans -hydroxylation ratio, and in increased shoot-to-root transport of cytokinins in the phloem. Phloem mobility and basipetal transport of cytokinins were also demonstrated by feeding zeatin riboside into the phloem. The resulting enrichment of cytokinins in the roots caused an increased expression of a highaffinity nitrate transporter, the enrichment of amino compounds (Glu, Val, Phe, Lys) in the fine roots and a significant decrease in nitrate net uptake. Significant enrichment in amino acids ‐ accompanied by decreased nitrate net uptake but not by increased expression of the highaffinity nitrate transporter ‐ was also observed when Gln the major long-distance transport form of nitrogen in beech was fed into the phloem. These results provide experimental evidence that, in addition to amino compounds, cytokinins that are known to cycle within the plant are also communicating changes in N metabolism from the shoot to the roots. Apparently increased cytokinin contents in the root can transiently increase nitrate uptake, but this upregulation may be overridden by transcriptional and posttranscriptional down-regulation mediated by amino compounds.

The fluxes of the greenhouse gases methane (CH4) and nitrous oxide (N2O) were measured in mangrove wetlands in Queensland, Australia, using the closed chamber technique. Large differences in the fluxes of both gases from different study sites were observed, which presumably depended on differences in substrate availability. CH4 emission rates were in the range of 20 to 350 μg m-2 h-1, whereas N2O fluxes were lower, amounting to - 2 to 14 μg m-2 h-1. In general, the field sites with high substrate availability showed higher emissions than sites with poor nutrient supply. This assumption is supported by the observation of dramatically increased N2O emissions (150 - 400 μg m-2 h-1) if study sites were artificially fertilised with additional N. As expected, N fertilisation did not alter CH4 fluxes during the period of investigation. In the present study, it was confirmed that the mangrove vegetation may play a role as a transport path for CH4 and N2O by facilitating diffusion out of the soil. Prop roots from Rhizophora stylosa emitted CH4 and N2O at rates of 2.6 and 3.3 μg m-2 root surface h-1, respectively, whereas the soil of this stand acted as a sink for CH4. As a consequence, the ecosystem as a whole could constitute a CH4 source despite CH4 uptake by the soil. In contrast to prop roots, the presence of pneumatophores in Avicennia marina led to a significant increase in CH4 emissions from mangrove soils, but did not enhance N2O emissions. These findings indicate that mangrove ecosystems may be considered a significant source of N2O and that anthropogenic nutrient input into these ecosystems will lead to enhanced source strengths. For an up-scaling of greenhouse gas emissions from mangrove forests to a global scale, more information is needed, particularly on the significance of vegetation.

At eight different dates during the 2000 growing season, d d d 13 C and d 18 O were determined in the phloem of adult beech trees growing in natural beech stands in south-west Germany differing in stand density and local climate. In addition, stand transpiration, precipitation, photosynthetic active radiation, relative air humidity, water pressure deficit of the air, air and soil temperature, soil water potential, and sugar concentration of the phloem sap were determined directly and evaporation and canopy stomatal conductance were modelled. All parameters were related to d 13 C. The study aimed to identify the time integral within which the d d d 13 C of organic compounds transported in the phloem is an indicative measure of these environmental influences. d 13 C of soluble carbon transported in the phloem was well correlated with mean stomatal conductance in a two-day integral prior to phloem sampling but did not depend on either light intensity or soil water availability. A strong positive relationship between d 13 C and d 18 O pointed to observed 13

The level of glutathione (GSH) in plants is important in defence reactions against biotic and abiotic stresses and can place considerable demand of the sulphur assimilation pathway. Enzymes involved in sulphur assimilation and GSH metabolism are not evenly distributed between different subcellular compartments or between different cell types in leaves or roots; however, there is little information on the effect that such asymmetries have on the actual GSH concentration in each compartment or cell type. In the present study in situ labelling with monochlorobimane (MCB) in combination with confocal laser scanning microscopy was used to quantify GSH in each of the main cell types of poplar leaves from fluorescence of the GSB conjugate formed. Comparison of results from the in situ approach with total GSH levels measured in vitro by highperformance liquid chromatography suggested that only the cytosolic GSH pool was labelled using this approach. This suggests that an appropriate GST was not present within the chloroplasts to catalyse the conjugation reaction and that chloroplastic GSH does not rapidly exchange with the cytoplasmic pool under the conditions of the assay. Cytosolic GSH levels were between 0.2 and 0.3 m M for both photosynthetic and non-photosynthetic (epidermal) cell types in wild-type poplar leaves. Cytosolic levels increased by around two-fold in transgenic poplars over-expressing bacterial g -glutamylsynthetase ( g g g -ECS) in the cytosol of all cell types, but there was no concomitant increase in the chloroplastic GSH pool.

Summary • Here, the effects were assessed of local climate and canopy thinning on the carbon isotope composition (δ13C) signatures of different plant groups in the understorey of a beech (Fagus sylvatica) forest in southern Germany. The relationship between δ13C and environmental parameters, within different time integrals, was also studied. • δ13C was analysed in different tissues of beech regeneration, and herbaceous and woody understorey vegetation, in thinned and untreated control stands differing in aspect and, hence, local climate, on three dates during the growing season. • Generally, tissues were 13C-depleted on the north-east, compared with the south-west aspect. Thinning had variable effects on δ13C, depending on plant group and sampling date. δ13C in beech leaves and roots in control stands was mostly influenced by mean soil water potential in 4- and 8-wk integrals before sampling, respectively, and in leaves, additionally, by mean radiation in a 4-wk time integral. Shoot water potential and transpiration influenced foliar δ13C of beech whereas δ13C in the beech wood was modulated by soil temperature integrated over a 4-wk period before sampling. Above-ground tissues of woody and herbaceous plants were 13C-enriched in mid-summer; their δ13C was poorly related to environmental factors. • δ13C of various tissues of beech regeneration appears to be indicative of recent environmental conditions within the forest understorey and, consequently, this easy-to-determine physiological parameter could be used widely to assess effects of silvicultural treatments.

In the present field study with adult trees inhabited by Viscum album, the question was addressed as to whether European mistletoes are able to remove reduced sulphur from the xylem sap of its hosts. For this purpose the reduced sulphur composition and content of the xylem sap of Viscum album and the corresponding hosts Populus x euramericana and Abies alba were analysed. The xylem sap of Viscum was enriched in reduced sulphur compared to the hosts but still reflected the higher reduced sulphur content of Populus compared to Abies. Despite similar xylem sap composition of the hosts with glutathione as the dominating thiol, Viscum on Populus contained predominantly cysteine, Viscum on Abies predominantly glutathione in its xylem sap. These findings suggest selective and different removal of reduced sulphur from these hosts. Still the amount of reduced sulphur removed was too small to result in changes of the concentration of thiols in the xylem sap of the hosts that are statistically significant, probably due to the high variability encountered under field conditions. Despite the differences in the reduced sulphur composition and contents of the xylem sap between Viscum on Populus and Viscum on Abies, total thiol content as well as thiol composition of Viscum leaves on the two hosts were similar throughout the seasons. The seasonal pattern in the thiol composition and contents of Viscum leaves showed high levels in spring and autumn and low levels in summer. The significance of these seasonal changes is discussed.

Summary • The effects of N-availability and elevated atmospheric CO 2 partial pressure ( pCO2) on growth, allometry and N-metabolism of poplar plants are reported here. • Poplar plants were grown hydroponically at deficient and sufficient N-supply under ambient and elevated pCO2. The N-fluxes within the plants were estimated by comparing the fate of newly acquired 15N-NO3− in plants either severely N-limited or with sufficient N-supply. • At deficient N-supply, plants accumulated less biomass and exhibited an increased root : shoot ratio compared with sufficient N-supply; a larger fraction of newly acquired 15 N was allocated to the youngest leaves immediately after exchange of the nutrient solution. Increasing the external N-supply from deficient to sufficient shifted the site of nitrate reduction from roots to leaves. • Elevated pCO2 increased total biomass and the root : shoot ratio at deficient N-supply, but had no effect at sufficient N-supply. Elevated pCO2 decreased rates of N-uptake in both treatments. Increased root : shoot ratio at deficient N-supply coincided with enhanced nitrate reduction in the root and elevated pCO2 also enhanced the allocation of newly acquired 15N to the youngest leaves. Root nitrate reduction as a possible factor controlling the root : shoot ratio and N-allocation is discussed.

The aim of the present study was to analyse whether offspring of mature Quercus ilex trees grown under life-long elevated pCO2 show alterations in the physiological response to elevated pCO2 in comparison with those originating from mature trees grown at current ambient pCO2. To investigate changes in C- (for changes in photosynthesis, biomass and lignin see Polle, McKee & Blaschke Plant, Cell and Environment 24, 1075–1083, 2001), N-, and S-metabolism soluble sugar, soluble non-proteinogenic nitrogen compounds (TSNN), nitrate reductase (NR), thiols, adenosine 5′-phosphosulphate (APS) reductase, and anions were analysed. For this purpose Q. ilex seedlings were grown from acorns of mother tree stands at a natural spring site (elevated pCO2) and a control site (ambient pCO2) of the Laiatico spring, Central Italy. Short-term elevated pCO2 exposure of the offspring of control oaks lead to higher sugar contents in stem tissues, to a reduced TSNN content in leaves, and basipetal stem tissues, to diminished thiol contents in all tissues analysed, and to reduced APS reductase activity in both, leaves and roots. Most of the components of C-, N- and S-metabolism including APS reductase activity which were reduced due to short-term elevated pCO2 exposure were recovered by life-long growth under elevated pCO2 in the offspring of spring oaks. Still TSNN contents in phloem exudates increased, nitrate contents in lateral roots and glutathione in leaves and phloem exudates remained reduced in these plants. The present results demonstrated that metabolic adaptations of Q. ilex mother trees to elevated pCO2 can be passed to the next generation. Short- and long-term effects on source-to-sink relation and physiological and genetic acclimation to elevated pCO2 are discussed.

Recently, phytoremediation of soils polluted with heavy metals has received a lot of attention. Since glutathione (GSH) and its derivatives (e.g., phytochelatins) play a major role in plant defence against environmental pollutants, we tested the effects of over-expression of bacterial genes for GSH synthesis in poplar on cadmium accumulation. A pilot experiment with CdCl2 in hydroponics revealed that poplars over-expressing γ-glutamylcysteine synthetase (γ-ECS) accumulated significantly more Cd in root tissue than wild type or glutathione synthetase over-expressing poplars. To test the partitioning of Cd in different organs, poplar lines over-expressing γ-ECS in the cytosol and in chloroplasts were treated with 0.2 mM CdCl2 in hydroponics. Significant amounts of Cd were translocated to leaves, but significant differences in Cd accumulation were not observed between transgenic and wild type plants. To evaluate these lines for large-scale phytoremediation of cadmium, plants were treated with 2 mM Cd in soil. Over a four-week period, the poplar plants were able to accumulate up to 5.3 mg Cd. Most remarkably, in young leaves of both transgenic lines, Cd was accumulated to concentrations 2.5 - 3 times higher than in the wild type. The increased allocation of cadmium to the young leaves represents a potentional advantage for the phytoremediation process using the same plants over several vegetation periods. The use of transgenic poplar lines with enhanced glutathione production capacity seems to be of particular advantage in highly polluted soils.