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Key message Sessile oak leaves showed a high degree of plasticity to atmospheric and pedospheric conditions. Abstract The aim of the present study was to elucidate the significance of current weather conditions for foliar traits of adult sessile oak (Quercus petraea), one of the most valuable forest tree species in Central Europe. For this purpose, structural and functional traits were analysed in fully expanded, sun exposed leaves collected in south-west Germany from five old-growth forest stands, representing the meteorological and pedospheric conditions in the growing region, but differing in aridity during the 12 days before harvest in two consecutive years. Across the forest stands, most foliar traits differed significantly between wet and dry weather conditions before harvest as indicated by partial least square discriminant analysis (PLS-DA). These traits included fresh weight/dry weight ratio, leaf hydration, leaf-C content, leaf-C/N ratio, structural N, soluble protein-N, total amino acid-N, cell wall composition, numerous specific amino acids as well as soluble sugar content. Structural biomass, δ13C signature, total N and total C as well as H2O2 contents were not affected by the weather before harvest. These results indicate a high plasticity of the foliar metabolism of drought-tolerant sessile oak to current weather conditions. They also suggest that sessile oak is characterized by a high potential to cope with the growth conditions expected as a consequence of future climate change.

Nitrogen (N) loading and extreme drought were found to strongly alter biomass production, species composition, carbon and water fluxes of temperate grasslands. Such changes at the community level are often attributed to species and functional group‐specific responses in phenology and/or physiology. In a multifactorial field experiment, we studied the responses of three abundant grassland species (forb Centaurea jacea, grasses Arrhenatherum elatius and Dactylis glomerata) to N loading and extreme drought, focusing on responses in carbon and water relations at the leaf level. We analyzed 1) changes in bulk leaf N (uptake efficiency of additional N), 2) adaptation of plant water status (leaf water potential) and 3) the impact on leaf carbon and water fluxes. We observed more efficient N utilization in both grasses compared to C. jacea. Naturally occurring summer drought impacted the plant water status of all species significantly while extreme drought treatment only affected water status additionally during and after summer drought. C. jacea was able to maintain much lower leaf water potentials compared to grasses during drought. Despite these clear species‐specific responses to N loading and drought in bulk, species were able to maintain homeostasis of leaf carbon and water fluxes. Thus, strong declines in the (community) carbon sequestration observed at this site during the (natural) summer drought were not related to leaf physiological responses in assimilation but driven by phenological adaptions of the species community: The drought sensitive grasses, even though exhibiting a higher N uptake efficiency, responded with a shortened life cycle to severe summer drought.

Due to climate change, sessile oak (Quercus petraea) seedlings experience an increasing risk of drought during regeneration of forest stands by management practices. The present study was aimed at elucidating the potential of sessile oak seedlings originating from sites with different aridity and nitrogen (N) supply to acclimate to contrasting water availability. For this purpose, a free-air cross-exchange experiment was conducted between a dry and a humid forest stand with high and low soil N contents, respectively, during two consecutive years differing in aridity before harvest. Almost all structural and physiological foliar traits analyzed did not differ consistently between seed origins during both years, when cultivated at the same site. As an exception, the arid provenance upregulated foliar ascorbate contents under drought, whereas the humid provenance accumulated the phenolic antioxidants vescalagin and castalagin (VC) under favorable weather conditions and consumed VC upon drought. Apparently, differences in long-term aridity at the forest sites resulted in only few genetically fixed differences in foliar traits between the provenances. However, structural and physiological traits strongly responded to soil N contents and weather conditions before harvest. Foliar N contents and their partitioning were mostly determined by the differences in soil N availability at the sites, but still were modulated by weather conditions before harvest. In the first year, differences in aridity before harvest resulted in differences between most foliar traits. In the second year, when weather conditions at both sites were considerably similar and more arid compared to the first year, differences in foliar traits were almost negligible. This pattern was observed irrespective of seed origin. These results support the view that leaves of sessile oak seedlings generally possess a high plasticity to cope with extreme differences in aridity by immediate acclimation responses that are even better developed in plants of arid origin.

Climate change, specifically the increasing frequency and intensity of summer heat and drought, has severe influences on the performance of beech forests, including decline in growth, reduced nutrient turnover, enhanced mortality, and a shift in spatial distribution northwards and towards higher elevations. The present study aimed to characterize the physiological responses of Croatian beech saplings originating from 10 natural forest stands to experimentally applied water deprivation in a common-garden experiment. The aim was to evaluate the extent to which external factors such as climate, as well as nitrogen (N) and phosphorus (P) availability in the soil of the natural habitats, control the response of beech saplings to water deprivation. For this purpose, beech saplings from 10 forest stands that differed in terms of soil type, chemical soil properties, as well as climate were collected in winter, cultivated in an artificial soil substrate under controlled conditions for one year, and then subjected to 29 days of water deprivation. Responses to water deprivation were observed in the antioxidative system (total ascorbate, reduced ascorbate, oxidized ascorbate, and redox state) in leaves and fine roots. The latter allowed us to categorize saplings as adapted or sensitive to water deprivation. P over N availability in the soil rather than climatic conditions in the natural habitats controlled the response of beech saplings to the water-deprivation event. The categorization of saplings as adapted or sensitive to water deprivation was related to genetic parameters. The results of this multidisciplinary study (tree physiology, climate, and genetic data) are considered to be highly significant and beneficial for the adaptation of European beech forests to changing climatic conditions.

The interplay of the processes controlling carbon partitioning into plant primary and secondary metabolisms, such as respiratory CO2 release and Volatile Organic Compound (VOC) biosynthesis, is still not fully understood. Pyruvate is a key metabolite which is formed in primary metabolism and acting as substrate in numerous secondary pathways forming many BVOCs, such as isoprene, volatile terpenoids, oxygenated compounds (acetaldehyde, acetic acid), benzenoids and fatty acid oxidation products (several wound VOCs), which can be emitted by plants. Within the ERC project VOCO, we established an innovative analytical setup enabling us to simultaneously measure stable carbon isotope composition of leaf exchanged CO2 (Infrared Isotope spectroscopy IRIS) together with VOC release (PTR-TOF-MS and GC-MS-C-IRMS). Position specific 13C-labeled pyruvate and 13CO2 were applied in tracer experiments to elucidate carbon partitioning at metabolic branching points into VOCs vs. CO2 in drought stressed Scots pine trees.We observed treatment specific patterns of VOC emission including volatile terpenoids, oxygenated BVOCs and green leaf volatiles. Tracing 13C, we elucidated if compounds were de novo synthesized from 13C-labeled pyruvate or 13CO2. Position-specific labeling with [1-13C]-pyruvate and [2-13C]-pyruvate suggested that most VOCs were synthesized from the C2-C3 moiety of pyruvate whereas the C1 position was decarboxylated resulting in 13CO2 release by dark respiration in the light. We observed drought stress related shifts in 13CO2 release and VOC emissions. Our observations suggest that VOC emissions are associated with significant pyruvate C1 decarboxylation which is released to the atmosphere. This novel approach contributes to a better understanding of the metabolic links of processes of primary and secondary plant metabolisms which is relevant for the vegetation-atmosphere exchange of CO2 and VOCs.

Atmospheric p CO 2 impacts Quercus petraea biomass production and cell wall composition of the leaves in favor of cellulose at the expense of lignin, and enhances foliar non-structural carbohydrate levels and sucrose contents in a pCO 2 concentration-dependent manner. Sessile oak (Quercus petraea Liebl.) was grown for ca. half a year from seeds at ambient control (525 ppm), 750, 900, and 1000 ppm atmospheric pCO2 under controlled conditions. Increasing pCO2 enhanced biomass production, modified the cell wall composition of the leaves in favor of cellulose at the expense of lignin, and enhanced the foliar non-structural carbohydrate level, in particular the sucrose content; as well as total N content of leaves by increased levels of all major N fractions, i.e., soluble proteins, total amino acids, and structural N. The enhanced total amino acid level was largely due to 2-ketoglutarate and oxalo acetate-derived compounds. Increasing pCO2 alleviated oxidative stress in the leaves as indicated by reduced H2O2 contents. High in vitro glutathione reductase activity at reduced H2O2 contents suggests enhanced ROS scavenging, but increased lipid peroxidation may also have contributed, as indicated by a negative correlation between malone dialdehyde and H2O2 contents. Almost all these effects were at least partially reversed, when pCO2 exceeded 750 or 900 ppm. Apparently, the interaction of atmospheric pCO2 with leaf structural and physiological traits of Q. petraea seedlings is characterized by a dynamic response depending on the pCO2 level.

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.

The Venus flytrap Dionaea muscipula captures insects and consumes their flesh. Prey contacting touch-sensitive hairs trigger traveling electrical waves. These action potentials (APs) cause rapid closure of the trap and activate secretory functions of glands, which cover its inner surface. Such prey-induced haptoelectric stimulation activates the touch hormone jasmonate (JA) signaling pathway, which initiates secretion of an acidic hydrolase mixture to decompose the victim and acquire the animal nutrients. Although postulated since Darwin’s pioneering studies, these secretory events have not been recorded so far. Using advanced analytical and imaging techniques, such as vibrating ion-selective electrodes, carbon fiber amperometry, and magnetic resonance imaging, we monitored stimulus-coupled glandular secretion into the flytrap. Trigger-hair bending or direct application of JA caused a quantal release of oxidizable material from gland cells monitored as distinct amperometric spikes. Spikes reminiscent of exocytotic events in secretory animal cells progressively increased in frequency, reaching steady state 1 d after stimulation. Our data indicate that trigger-hair mechanical stimulation evokes APs. Gland cells translate APs into touch-inducible JA signaling that promotes the formation of secretory vesicles. Early vesicles loaded with H+ and Cl− fuse with the plasma membrane, hyperacidifying the "green stomach"-like digestive organ, whereas subsequent ones carry hydrolases and nutrient transporters, together with a glutathione redox moiety, which is likely to act as the major detected compound in amperometry. Hence, when glands perceive the haptoelectrical stimulation, secretory vesicles are tailored to be released in a sequence that optimizes digestion of the captured animal.

European mistletoe (Viscum album L.) has largely infested Central European forests and causes high mortality probability particularly in dry years. However, little information is available about the consequences of mistletoe infestation for metabolic processes in bark and wood of its host, despite their important roles in infestation defense. We analyzed the tissue hydration, carbohydrate composition, phytohormone profile, reactive oxygen species and anti-oxidant levels in bark and wood of Scots pines (Pinus sylvestris L.), as dependent on mistletoe infestation. As a consequence of mistletoe infestation, host bark and wood showed impaired hydration and reduced total carbon content. In the bark, soluble sugar and lignin contents increased, apparently at the expense of holo-cellulose. Hydrogen peroxide accumulation was accompanied by increased glutathione and decreased reduced ascorbic acid levels. Mistletoe infestation mediated alteration of the phytohormone profile in bark and wood of its host. Cytokinins, jasmonic acid and abscisic acid levels increased in both tissues, whereas salicylic acid and indole-3-acetic acid, which were only detected in the bark, declined. The present results show that mistletoe infestation affects both the host's anti-oxidative defense system and the phytohormone profile after establishment of the xylem tapping haustorium. The significance of these processes for the development of the woody mistletoe stem and the haustorium is discussed.

Atmospheric p CO

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 H

Clones of Norway spruce (Picea abies L.) were grown for several years on an altitudinal gradient (1750 m, 1150 m and 800 m above sea level) to study the effects of environmental × genetic interactions on growth and foliar metabolites (protein, pigments, antioxidants). Clones at the tree line showed 4.3-fold lower growth rates and contained 60% less chlorophyll (per gram of dry matter) than those at valley level. The extent of growth reduction was clone-dependent. The mortality of the clones was low and not altitude-dependent. At valley level, but not at high altitude, needles of mature spruce trees showed lower pigment and protein concentrations than clones. In general, antioxidative systems in needles of the mature trees and young clones did not increase with increasing altitude. Needles of all trees at high altitude showed higher concentrations of dehydroascorbate than at lower altitudes, indicating higher oxidative stress. In one clone, previously identified as sensitive to acute ozone doses, this increase was significantly higher and the growth reduction was stronger than in the other genotypes. This clone also displayed a significant reduction in glutathione reductase activity at high altitude. These results suggest that induction of antioxidative systems is apparently not a general prerequisite to cope with altitude in clones whose mother plants originated from higher altitudes (about 650-1100 m above sea level, Hercycnic-Carpathian distribution area), but that the genetic constitution for maintenance of high antioxidative protection is important for stress compensation at the tree line.

The critical level for SO 2 susceptibility of Populus × canescens is approximately 1.2 μL L −1 SO 2 . Both sulfite oxidation and sulfite reduction and assimilation contribute to SO 2 detoxification. In the present study, uptake, susceptibility and metabolism of SO2 were analyzed in the deciduous tree species poplar (Populus × canescens). A particular focus was on the significance of sulfite oxidase (SO) for sulfite detoxification, as SO has been characterized as a safety valve for SO2 detoxification in herbaceous plants. For this purpose, poplar plants were exposed to different levels of SO2 (0.65, 0.8, 1.0, 1.2 μL L−1) and were characterized by visible injuries and at the physiological level. Gas exchange parameters (stomatal conductance for water vapor, CO2 assimilation, SO2 uptake) of the shoots were compared with metabolite levels (sulfate, thiols) and enzyme activities [SO, adenosine 5′-phosphosulfate reductase (APR)] in expanding leaves (80–90 % expanded). The critical dosage of SO2 that confers injury to the leaves was 1.2 μL L−1 SO2. The observed increase in sulfur containing compounds (sulfate and thiols) in the expanding leaves strongly correlated with total SO2 uptake of the plant shoot, whereas SO2 uptake rate was strongly correlated with stomatal conductance for water vapor. Furthermore, exposure to high concentration of SO2 revealed channeling of sulfite through assimilatory sulfate reduction that contributes in addition to SO-mediated sulfite oxidation to sulfite detoxification in expanding leaves of this woody plant species.

Glutathione is involved in thiol redox signaling and acts as a major redox buffer against reactive oxygen species, helping to maintain a reducing environment in vivo. Glutathione reductase (GR) catalyzes the reduction of glutathione disulfide (GSSG) into reduced glutathione (GSH). The Arabidopsis thaliana genome encodes two GRs: GR1 and GR2. Whereas the cytosolic/peroxisomal GR1 is not crucial for plant development, we show here that the plastid-localized GR2 is essential for root growth and root apical meristem (RAM) maintenance. We identify a GR2 mutant, miao, that displays strong inhibition of root growth and severe defects in the RAM, with GR activity being reduced to ∼50%. miao accumulates high levels of GSSG and exhibits increased glutathione oxidation. The exogenous application of GSH or the thiol-reducing agent DTT can rescue the root phenotype of miao, demonstrating that the RAM defects in miao are triggered by glutathione oxidation. Our in silico analysis of public microarray data shows that auxin and glutathione redox signaling generally act independently at the transcriptional level. We propose that glutathione redox status is essential for RAM maintenance through both auxin/PLETHORA (PLT)-dependent and auxin/PLT-independent redox signaling pathways.

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.

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).

The poplar hybrid Populus x canescens (syn. Populus tremula x Populus alba) was subjected to salt stress by applying 75 mM NaCl for 2 weeks in hydroponic cultures. Decreasing maximum quantum yield (Fv/Fm) indicated damage of photosystem II (PS II), which was more pronounced under nitrate compared with ammonium nutrition. In vivo staining with diaminobenzidine showed no accumulation of H(2)O(2) in the leaf lamina; moreover, staining intensity even decreased. But at the leaf margins, development of necrotic tissue was associated with a strong accumulation of H(2)O(2). Glutathione (GSH) contents increased in response to NaCl stress in leaves but not in roots, the primary site of salt exposure. The increasing leaf GSH concentrations correlated with stress-induced decreases in transpiration and net CO(2) assimilation rates at light saturation. Enhanced rates of photorespiration could also be involved in preventing reactive oxygen species formation in chloroplasts and, thus, in protecting PS II from damage. Accumulation of Gly and Ser in leaves indeed indicates increasing rates of photorespiration. Since Ser and Gly are both immediate precursors of GSH that can limit GSH synthesis, it is concluded that the salt-induced accumulation of leaf GSH results from enhanced photorespiration and is thus probably restricted to the cytosol.

The coniferous forest tree Douglas-fir (Pseudotsuga menziesii) is native to the pacific North America, and is increasingly planted in temperate regions worldwide. Nitrogen (N) metabolism is of great importance for growth, resistance and resilience of trees. In the present study, foliar N metabolism of adult trees of three coastal and one interior provenance of Douglas-fir grown at two common gardens in southwestern Germany (Wiesloch, W; Schluchsee, S) were characterized in two subsequent years. Both the native North American habitats of the seed sources and the common garden sites in Germany differ in climate conditions. Total and mineral soil N as well as soil water content were higher in S compared to W. We hypothesized that i) provenances differ constitutively in N pool sizes and composition, ii) N pools are affected by environmental conditions, and iii) that effects of environmental factors on N pools differ among interior and coastal provenances. Soil water content strongly affected the concentrations of total N, soluble protein, total amino acids (TAA), arginine and glutamate. Foliar concentrations of total N, soluble protein, structural N and TAA of trees grown at W were much higher than in trees at S. Provenance effects were small but significant for total N and soluble protein content (interior provenance showed lowest concentrations), as well as arginine, asparagine and glutamate. Our data suggest that needle N status of adult Douglas-fir is independent from soil N availability and that low soil water availability induces a re-allocation of N from structural N to metabolic N pools. Small provenance effects on N pools suggest that local adaptation of Douglas-fir is not dominated by N conditions at the native habitats.

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 a field study, the composition and concentrations of amino compounds in the xylem sap of the mistletoe, Viscum album L., and in the xylem sap of two host species, an evergreen conifer (Abies alba Mill.) and a deciduous broad-leaved tree (Populus x euramericana), were analyzed. The xylem sap of both hosts and mistletoe contained large, but similar amounts of total organic nitrogen in low molecular weight amino compounds (TONLW). Nevertheless, individual amino compounds accumulated in the xylem sap of mistletoe relative to the host xylem sap, indicating selective uptake. In the xylem sap of Populus, major amino compounds (asparagine (Asn) and glutamine (Gln)) and the bulk parameters, TONLW and proteinogenic amino acids, showed significant seasonal variation. In Abies and in mistletoe on either host, variation of amino compounds in xylem sap was largely explained by inter-annual differences, not by seasonal variation. In both hosts, TONLW in the xylem sap was dominated by Gln. There was a steady decrease in relative abundance of Gln from the host xylem sap to the mistletoe xylem sap and to the stems and leaves of mistletoe. Simultaneously, the abundance of arginine (Arg) increased. Arginine was the predominant amino compound in the stems and leaves of mistletoe, occurring at concentrations previously observed only in leaves of trees exposed to excess nitrogen. We conclude that Gln (2 mol N mol(-1)) delivered by the host xylem sap is converted, in mistletoe, to Arg (4 mol N mol(-1)) and that the organic carbon liberated from Gln contributes significantly to the parasite's heterotrophic carbon gain. Statistical analyses of the data support this conclusion. Accumulation of Arg in mistletoe is an indication of excess N supply as a result of the uptake of amino compounds from the host xylem sap and a lack of phloem uploading.

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.

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.

Wild-type hybrid poplar (Populus tremula × P. alba) plants and transgenic lines over-expressing glutathione reductase (GR) either in the cytosol (ca. 5-fold) or in the chloroplast (150- to 200-fold) or glutathione synthetase (GSS) in the cytosol (ca. 200-fold) were exposed for 3 days to ambient air or air containing 300 nl l–1 ozone for 7 h day–1. The contents and oxidation state of antioxidants (glutathione; ascorbate), the in vitro activities of target enzymes (GR, GSS), as well as the in vitro activities of ascorbate consuming (ascorbate peroxidase, APX) and regenerating (monodehydroascorbate reductase, MDAR; dehydroascorbate reductase, DHAR) enzymes were determined in the developing 2nd and the mature 5th leaves from the apex of the plants. The changes in the activities of the target enzymes GR and GSS also affected other components of the leaves' antioxidative system. Over-expression of GR in the chloroplast enhanced total glutathione contents of the leaves 2- to 3-fold. Foliar GSSG levels were enhanced in all transgenic poplar lines compared to wild-type plants. In developing leaves of transgenic plants, over-expressing GR in the cytosol or chloroplasts, APX and DHAR activities were higher, in mature leaves similar or lower than in wild-type plants. These differences between poplar lines did not mediate different sensitivities of the leaves to acute ozone exposure (Strohm et al., J Exp Bot 50:365–374). Still, components of the antioxidative system of the leaves showed both similar or specific reactions to acute ozone exposure in different poplar lines. Irrespective of leaf age total glutathione contents increased 2- to 3-fold in ozone-exposed leaves of all poplar lines. Also GSSG contents of the leaves increased, with the exception of plants over-expressing GR in the chloroplasts. Still a highly reduced state of glutathione was maintained in wild-type (>90%) and transgenic lines (ca. 80%) irrespective of ozone exposure. Apparently, GR activity was sufficient in all lines to compensate for ozone-mediated glutathione oxidation. Foliar GR activity increased up to 100% as a consequence of ozone treatment in wild-type and transgenic plants over-expressing GR in the cytosol, but not in plants over-expressing GR in the chloroplasts. The combined increase in total glutathione contents and GR activity in response to acute ozone exposure did not prevent injury and, therefore, cannot be considered a useful mechanism of defence, but rather an indicator of ozone-mediated oxidative stress. Total ascorbate contents and the activities of ascorbate consuming (APX) or regenerating enzymes (MDAR, DHAR) were not significantly affected by acute ozone exposure in all poplar lines. Ozone exposure strongly oxidised the foliar ascorbate pool except in leaves over-expressing GR in the chloroplast. Apparently, these leaves possess an enhanced capacity for ascorbate regeneration as a consequence of the strongly enhanced chloroplast GR activity and the simultaneously increased APX activity and glutathione levels. The enhanced antioxidative capacity did not prevent ozone-mediated injury to the leaves, most likely due to different sites of primary ozone reactions. Despite higher total ascorbate contents and higher APX activities in developing than in mature leaves, these and other components of the antioxidative system analysed in this study were not responsible for the higher sensitivity to acute ozone exposure of mature compared to developing poplar leaves.

Global climate change causes an increase in ambient air temperature, a major environmental factor influencing plant physiol-ogy and growth that already has been perceived at the regional scale and is expected to become even more severe in the future. In the present study, we investigated the effect of elevated ambient air temperature on the nitrogen metabolism of two interior provenances of Douglas fir (Pseudotsuga menziesii var. glauca) originating from contrasting habitats, namely the provenances Monte Creek (MC) from a drier environment and Pend Oreille (PO) from a more humid environment. Three- to four-year-old seedlings of the two provenances were grown for 3 months in controlled environments under either control temperature (day 20 °C, night 15 °C) or high temperature (HT, 30/25 °C) conditions. Total nitrogen (N), soluble protein, chlo-rophyll and total amino acid (TAA) contents as well as individual amino acid concentrations were determined in both current-year and previous-year needles. Our results show that the foliar total N contents of the two provenances were unaffected by HT. Arginine, lysine, proline, glutamate and glutamine were the most abundant amino acids, which together contributed 88% to the TAA pool of current- and previous-year needles. High temperature decreased the contents of most amino acids of the glutamate family (i.e., arginine, proline, ornithine and glutamine) in current-year needles. However, HT did not affect the concentrations of metabolites related to the photorespiratory pathway, such as NH4+, glycine and serine. In general, current-year needles were considerably more sensitive to HT than previous-year needles. Moreover, provenance PO originating from a mesic environment showed stronger responses to HT than provenance MC. Our results indicate provenance-specific plasticity in the response of Douglas fir to growth temperature. Provenance-specific effects of elevated temperature on N-use efficiency suggest that origin might determine the sensitivity and growth potential of Douglas fir trees in a future warmer climate. Global climate change causes an increase in ambient air temperature, a major environmental factor influencing plant physiology and growth that already has been perceived at the regional scale and is expected to become even more severe in the future. In the present study, we investigated the effect of elevated ambient air temperature on the nitrogen metabolism of two interior provenances of Douglas fir (Pseudotsuga menziesii var. glauca) originating from contrasting habitats, namely the provenances Monte Creek (MC) from a drier environment and Pend Oreille (PO) from a more humid environment. Three- to four-year-old seedlings of the two provenances were grown for 3 months in controlled environments under either control temperature (day 20 °C, night 15 °C) or high temperature (HT, 30/25 °C) conditions. Total nitrogen (N), soluble protein, chlorophyll and total amino acid (TAA) contents as well as individual amino acid concentrations were determined in both current-year and previous-year needles. Our results show that the foliar total N contents of the two provenances were unaffected by HT. Arginine, lysine, proline, glutamate and glutamine were the most abundant amino acids, which together contributed ∼88% to the TAA pool of current- and previous-year needles. High temperature decreased the contents of most amino acids of the glutamate family (i.e., arginine, proline, ornithine and glutamine) in current-year needles. However, HT did not affect the concentrations of metabolites related to the photorespiratory pathway, such as NH+4, glycine and serine. In general, current-year needles were considerably more sensitive to HT than previous-year needles. Moreover, provenance PO originating from a mesic environment showed stronger responses to HT than provenance MC. Our results indicate provenance-specific plasticity in the response of Douglas fir to growth temperature. Provenance-specific effects of elevated temperature on N-use efficiency suggest that origin might determine the sensitivity and growth potential of Douglas fir trees in a future warmer climate.

summary 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. ev. Wembley) was grown from seed, in controlled-environment chambers, under reciprocal combinations of [CO2] at 350 or 700 μmol mol−1and [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.

A decreased utilization of NADPH for CO2 fixation as a result of carbohydrate accumulation in chlorotic leaves is generally believed to be associated with an increase in oxidative stress. Molecular oxygen may serve as an alternative electron acceptor of photosynthesis under these conditions. In order to test this hypothesis mature leaves of tobacco plants (Nicotiana tabacum) were detached, fed with glucose (50 mM) via the petiole, and used to study the effect of carbohydrate accumulation on the pigment content and the components of the superoxide dismutase-ascorbate-glutathione cycle. During a period of five days the concentration of total soluble carbohydrates increased substantially in leaves supplied with glucose in comparison with control leaves supplied only with water. This increase was accompanied by a twofold decrease in the chlorophyll content. In detached water-fed leaves the levels of most of the antioxidative components increased, whereas glucose feeding had no or only little additional effect on the activities of the protective enzymes, but caused a 1.6- and 4-fold increase in the contents of glutathione and ascorbate, respectively. In relation to the total foliar ascorbate pool, the amount of reduced ascorbate increased from about 30 % to 60 % upon feeding with glucose. These results do not support the idea that the accumulation carbohydrates per se cause an increased rate of superoxide production which necessitates increased activities of antioxidative enzymes. It rather appears that glucose-fed leaves have an increased reductive capacity that can be released via glutathione into the ascorbate pool, thereby, regulating the redox state of ascorbate.

summary Needles from young and mature Scots pine trees (Pinus sylvestirs) were collected at three field sites in eastern Germany, i.e. Menz, Taura, and Gossa, which differed in SO2 pollution. Current and previous year's needles from the polluted sites Taura and Gossa (yearly mean af SO2: 26 ppb) contained approximately 100-120 μmol sulphate g −1 d. wt, whereas needles tram the unpolluted site, Menz (yearly mean of SO2: 3 ppb) contained 30-65 μmol sulphate g−1 d. wt. The apoplastic sulphate content was in the order of o.3-1% of total sulphate and corresponded to concentrations of 0.6-1.2 mM in needles from Mtnz and to 1.5–2 mn in needles from Gossa. In needles from the polluted sites the contents of glutathione, cysteine, γ glutamyleysteine, and soluble protein were higher than in needles from Menz. Despite the clear increases in sulphur-containing compounds, pigment content and activities of superoxide dismutase, total peroxidase and apoplastic peroxidase (on the basis of d. wt) were not significantly different. When enzymatic activities were related to protein, lowest activities were found in needles from the site with the highest SO2-pollution. The results show that it is not possible to use the activities of peroxidases or superoxide dismutases as biomarkers for SO2-mediated stress in pine needles. It remains open whether the needles from the polluted sites suffered from greater oxidative stress than the needles from the less polluted site.

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).

Drought is a major environmental stress affecting growth and vitality of forest ecosystems. In the present study, foliar nitrogen (N) and carbon (C) metabolism of two Douglas fir (Pseudotsuga menziesii) provenances with assumed different drought tolerance were investigated. We worked with 1-year-old seedlings of the interior provenance Fehr Lake (FEHR) originating from a dry environment and the coastal provenance Snoqualmie (SNO) from a more humid origin. Total C and N, structural N and the concentrations of soluble protein, total amino acids (TAAs) and individual amino acids as well as the relative abundance of polar, low-molecular-weight metabolites including antioxidants were determined in current-year needles exposed either to 42 days of drought or to 42 days drought plus 14 days of rewatering. The seedlings reacted in a provenance-specific manner to drought stress. Coastal provenance SNO showed considerably increased contents of TAAs, which were caused by increased abundance of the quantitatively most important amino acids arginine, ornithine and lysine. Additionally, the polyamine putrescine accumulated exclusively in drought-stressed trees of this provenance. In contrast, the interior provenance FEHR showed the opposite response, i.e., drastically reduced concentrations of these amino acids. However, FEHR showed considerably increased contents of pyruvate-derived and aromatic amino acids, and also higher drought-induced levels of the antioxidants ascorbate and α-tocopherol. In response to drought, both provenances produced large amounts of carbohydrates, such as glucose and fructose, most likely as osmolytes that can readily be metabolized for protection against osmotic stress. We conclude that FEHR and SNO cope with drought stress in a provenance-specific manner: the coastal provenance SNO was mainly synthesizing N-based osmolytes, a reaction not observed in the interior provenance FEHR; instead, the latter increased the levels of scavengers of reactive oxygen species. Our results underline the importance of provenance-specific reactions to abiotic stress.

The flux of inorganic and organic nitrogen into the mistletoe Viscum album L. from the xylem sap of a deciduous (Populus x euamericana) and a coniferous host (Abies alba Mill.) was analyzed. For this purpose, a perfusion system was developed in which the xylem sap of the host was replaced by an artificial perfusion solution. With this system flux rates into the mistletoe were determined in feeding experiments either with the organic nitrogen source [1,2-13C2]glutamine at high and the inorganic nitrogen source 15NO3- at low concentration or vice versa. Glutamine influx was already saturated at the low concentration in the xylem sap and was--different from nitrate--not enhanced, when a 250-fold higher concentration was applied. Nitrate influx matched glutamine influx only at high inorganic/organic nitrogen ratios in the perfusion solution. This result indicates a preferential influx of glutamine over nitrate from the host xylem into the mistletoe at the concentrations found in the xylem sap of trees. Surprisingly, a high percentage of both N sources were accumulated in the mistletoe stem, indicating excessive N nutrition of the mistletoe leaves. Since 13C isotope signature was significantly reduced in the outflowing perfusion solution, either an upload of organic compounds from the phloem into the xylem, or an efflux of organic compounds from haustorium of mistletoe into the xylem has to be assumed. 15N isotope signatures enriched in the outflowing perfusion solution support the idea of a nitrate uptake system at the host xylem-haustorium interface, which favors the light N isotope of nitrate.

Mixed spruce-beech plantations grown in large open-top chambers (OTC) were used to study consequences of elevated CO2, nitrogen-deposition and soil type on plant internal nitrogen and sulphur cycling of juvenile beech (Fagus sylvatica L.) and spruce (Picea abies Karst.) in a competitive situation. Processes of re-cycling as a consequence of protein turnover during leaf senescence in autumn were of further interest. For this purpose, phloem sap was collected in September 1998 and analysed for the composition and concentrations of organic and inorganic nitrogen and sulphur compounds. The phloem exudate of spruce showed higher total soluble non-protein nitrogen (TSNN) concentration on calcareous soil than on acidic soil, independent of the treatment. N-fertilization increased the N-concentration of phloem exudate significantly on both soil types, mainly by an increase of Arg and Gln concentrations. Elevated CO2 slightly increased TSNN on calcareous, but not on acidic soil. The combination of elevated CO2 and augmented N-deposition induced a further increase of TSNN on calcareous soil, but caused a lower N-effect on TSNN on acidic soil. Arg, the main TSNN component in phloem exudate, mediated this effect. Since Arg is considered to be a major nitrogen storage compound, it is concluded that in autumn elevated CO2 and augmented N-deposition, influence storage of N rather than N-supply of spruce. An effect of elevated CO2 and augmented N-deposition on GSH and sulphate concentrations in phloem exudate of spruce was not observed on acidic soil. On calcareous soil augmented N-deposition enhanced, elevated CO2 decreased phloem exudate GSH contents. In combination, elevated CO2 compensated the positive effect of N-deposition. The effects of elevated CO2 and augmented N-deposition on phloem sap N- and S-contents described above were not observed for beech trees. Apparently, elevated CO2 and augmented N-deposition did not affect plants internal S and N cycling of beech grown in spruce-beech plantations.

To study the role of low UV-B radiation in modulating the response of antioxidants to ozone, 4-year-old pine (Pinus sylvestris L.) and spruce (Picea abies L.) seedlings potted in natural soil, were exposed in phytochambers to fluctuating ozone concentrations between 9 and 113 nl I -1 according to field data recorded at Mt Wank (1175 m above sea level, Bavaria, Germany) and two-times ambient O 3 levels. UV-B radiation was either added at a biologically effective level of ca 1.2 kJ m -2 day -1 , which is close to that found in March at Mt Wank, or was excluded by filters (