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The growth of fruit trees depends on the nitrogen (N) remobilization in mature tissues and N acquisition from the soil. However, in evergreen mature citrus (Citrus reticulata Blanco) leaves, proteins with N storage functions and hub molecules involved in driving N remobilization remain largely unknown. Here, we combined proteome and physiological analyses to characterize the spatiotemporal mechanisms of growth of new leaves and storage protein degradation in mature leaves of citrus trees exposed to low-N and high-N fertilization in the field. Results show that the growth of new leaves is driven by remobilization of stored reserves, rather than N uptake by the roots. In this context, proline and arginine in mature leaves acted as N sources supporting the growth of new leaves in spring. Time-series analyses with gel electrophoresis and proteome analysis indicated that the mature autumn shoot leaves are probably the sites of storage protein synthesis, while the aspartic endopeptidase protein is related to the degradation of storage proteins in mature citrus leaves. Furthermore, bioinformatic analysis based on protein-protein interactions indicated that glutamate synthetase and ATP-citrate synthetase are hub proteins in N remobilization from mature citrus leaves. These results provide strong physiological data for seasonal optimization of N fertilizer application in citrus orchards., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Infection with the necrotrophic fungus Diplodia sapinea (Fr.) Fuckel is among the economically and ecologically most devastating diseases of conifers in the northern hemisphere and is accelerated by global climate change. This study aims to characterize the changes mediated by D. sapinea infection on its pine host (Pinus sylvestris L.) that lead to the death of its needles. For this purpose, we performed an indoor infection experiment and inoculated shoot tips of pine seedlings with virulent D. sapinea. The consequences for foliar traits, including the phytohormone profile, were characterized at both the metabolite and transcriptome level. Our results showed that D. sapinea infection strongly affected foliar levels of most phytohormones and impaired a multitude of other metabolic and structural foliar traits, such as reactive oxygen species scavenging. Transcriptome analysis revealed that these changes are partially mediated via modified gene expression by fungal exposure. Diplodia sapinea appears to overcome the defense reactions of its pine host by reprogramming gene expression and post-transcriptional controls that determine essential foliar metabolic traits such as the phytohormone profile, cell wall composition and antioxidative system., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Drought and salt exposure are among the most prevalent and severe abiotic stressors causing serious agricultural yield losses, alone and in combination. Little is known about differences and similarities in the effects of these two stress factors on plant metabolic regulation, particularly on nitrogen metabolism. Here, we studied the effects of water deprivation and salt exposure on water relations and nitrogen metabolites in leaves and roots of date palm seedlings. Both, water deprivation and salt exposure had no significant effects on plant water content or stable carbon (C) and nitrogen (N) isotope signatures. Significant effects of water deprivation on total C and N concentrations were only observed in roots, i.e., decreased total C and increased total N concentrations. Whereas salt exposure initially decreased total C and increased total N concentrations significantly in roots, foliar total C concentration was increased upon prolonged exposure. Initially C/N ratios declined in roots of plants from both treatments and upon prolonged salt exposure also in the leaves. Neither treatment affected soluble protein and structural N concentrations in leaves or roots, but resulted in the accumulation of most amino acids, except for glutamate and tryptophan, which remained stable, and serine, which decreased, in roots. Accumulation of the most abundant amino acids, lysine and proline, was observed in roots under both treatments, but in leaves only upon salt exposure. This finding indicates a similar role of these amino acids as compatible solutes in the roots in response to salt und drought, but not in the leaves. Upon prolonged treatment, amino acid concentrations returned to levels found in unstressed plants in leaves of water deprived, but not salt exposed, plants. The present results show both water deprivation and salt exposure strongly impact N metabolism of date palm seedlings, but in a different manner in leaves and roots., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Nitrogen (N) remobilization is an important physiological process that supports the growth and development of trees. However, in evergreen broad-leaved tree species, such as citrus, the mechanisms of N remobilization are not completely understood. Therefore, we quantified the potential of N remobilization from senescing leaves of spring shoots to mature leaves of autumn shoots of citrus trees under different soil N availabilities and further explored the underlying N metabolism characteristics by physiological, proteome and gene expression analyses. Citrus exposed to low N had an approximately 38% N remobilization efficiency (NRE), whereas citrus exposed to high N had an NRE efficiency of only 4.8%. Integrated physiological, proteomic and gene expression analyses showed that photosynthesis, N and carbohydrate metabolism interact with N remobilization. The improvement of N metabolism and photosynthesis, the accumulation of proline and arginine, and delayed degradation of storage protein in senescing leaves are the result of sufficient N supply and low N remobilization. Proteome further showed that energy generation proteins and glutamate synthase were hub proteins affecting N remobilization. In addition, N requirement of mature leaves is likely met by soil supply at high N nutrition, thereby resulting in low N remobilization. These results provide insight into N remobilization mechanisms of citrus that are of significance for N fertilizer management in orchards., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Drought negatively impacts growth and productivity of plants, particularly in arid and semi-arid regions. Although drought events can take place in summer and winter, differences in the impact of drought on physiological processes between seasons are largely unknown. The aim of this study was to elucidate metabolic strategies of date palms in response to drought in summer and winter season. To identify such differences, we exposed date palm seedlings to a drought-recovery regime, both in simulated summer and winter climate. Leaf hydration, carbon discrimination (${\Delta}$13C), and primary and secondary metabolite composition and contents were analyzed. Depending on season, drought differently affected physiological and biochemical traits of the leaves. In summer, drought induced significantly decreased leaf hydration, concentrations of ascorbate, most sugars, primary and secondary organic acids, as well as phenolic compounds, while thiol, amino acid, raffinose and individual fatty acid contents were increased compared with well-watered plants. In winter, drought had no effect on leaf hydration, ascorbate and fatty acids contents, but resulted in increased foliar thiol and amino acid levels as observed in summer. Compared with winter, foliar traits of plants exposed to drought in summer only partly recovered after re-watering. Memory effects on water relations, and primary and secondary metabolites seem to prepare foliar traits of date palms for repeated drought events in summer. Apparently, a well-orchestrated metabolic network, including the anti-oxidative system, compatible solutes accumulation and osmotic adjustment, and maintenance of cell-membrane stability strongly reduces the susceptibility of date palms to drought. These mechanisms of drought compensation may be more frequently required in summer., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

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., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Monosaccharide transporters (MSTs) represent key components of the carbon transport and partitioning mechanisms in plants, mediating the cell-to-cell and long-distance distribution of a wide variety of monosaccharides. In this study, we performed a thorough structural, molecular, and physiological characterization of the monosaccharide transporter gene family in the model legume Medicago truncatula. The complete set of MST family members was identified with a novel bioinformatic approach. Prolonged darkness was used as a test condition to identify the relevant transcriptomic and metabolic responses combining MST transcript profiling and metabolomic analysis. Our results suggest that MSTs play a pivotal role in the efficient partitioning and utilization of sugars, and possibly in the mechanisms of carbon remobilization in nodules upon photosynthate-limiting conditions, as nodules are forced to acquire a new role as a source of both C and N., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background and Aims: The processes connected with prey capture and the early consumption of prey by carnivorous Dionaea muscipula require high amounts of energy. The aim of the present study was to identify processes involved in flytrap energy provision and ATP homeostasis under these conditions., Methods: We determined photosynthetic CO2 uptake and chlorophyll fluorescence as well as the dynamics of ATP contents in the snap traps upon closure with and without prey., Key Results: The results indicate that upon prey capture, a transient switch from linear to cyclic electron transport mediates a support of ATP homeostasis. Beyond 4 h after prey capture, prey resources contribute to the traps' ATP pool and, 24 h after prey capture, export of prey-derived resources to other plant organs may become preferential and causes a decline in ATP contents., Conclusions: Apparently, the energy demand of the flytrap for prey digestion and nutrient mining builds on both internal and prey-derived resources., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Date palms are remarkably tolerant to environmental stresses, but the mechanisms involved remain poorly characterized. Leaf metabolome profiling was therefore performed on mature (ML) and young (YL) leaves of 2-year-old date palm seedlings that had been grown in climate chambers that simulate summer and winter conditions in eastern Saudi Arabia. Cultivation under high temperature (summer climate) resulted in higher YL H2O2 leaf levels despite increases in dehydroascorbate reductase (DHAR) activities. The levels of raffinose and galactinol, tricarboxylic acid cycle intermediates, and total amino acids were higher under these conditions, particularly in YL. The accumulation of unsaturated fatty acids, 9,12-octadecadienoic acid and 9,12,15-octadecatrienoic acid, was lower in ML. In contrast, the amounts of saturated tetradecanoic acid and heptadecanoic acid were increased in YL under summer climate conditions. The accumulation of phenolic compounds was favored under summer conditions, while flavonoids accumulated under lower temperature (winter climate) conditions. YL displayed stronger hydration, lower H2O2 levels, and more negative δ 13C values, indicating effective reactive oxygen species scavenging. These findings, which demonstrate the substantial metabolic adjustments that facilitate tolerance to the high temperatures in YL and ML, suggest that YL may be more responsive to climate change., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Plants close stomata when root water availability becomes limiting. Recent studies have demonstrated that soil-drying induces root-to-shoot sulfate transport via the xylem and that sulfate closes stomata. Here we provide evidence for a physiologically relevant signaling pathway that underlies sulfate-induced stomatal closure in Arabidopsis ( Arabidopsis thaliana ). We uncovered that, in the guard cells, sulfate activates NADPH oxidases to produce reactive oxygen species (ROS) and that this ROS induction is essential for sulfate-induced stomata closure. In line with the function of ROS as the second-messenger of abscisic acid (ABA) signaling, sulfate does not induce ROS in the ABA-synthesis mutant, aba3 - 1 , and sulfate-induced ROS were ineffective at closing stomata in the ABA-insensitive mutant abi2 - 1 and a SLOW ANION CHANNEL1 loss-of-function mutant. We provided direct evidence for sulfate-induced accumulation of ABA in the cytosol of guard cells by application of the ABAleon2.1 ABA sensor, the ABA signaling reporter ProRAB18 : GFP , and quantification of endogenous ABA marker genes. In concordance with previous studies, showing that ABA DEFICIENT3 uses Cys as the substrate for activation of the ABSCISIC ALDEHYDE OXIDASE3 (AAO3) enzyme catalyzing the last step of ABA production, we demonstrated that assimilation of sulfate into Cys is necessary for sulfate-induced stomatal closure and that sulfate-feeding or Cys-feeding induces transcription of NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3, limiting the synthesis of the AAO3 substrate. Consequently, Cys synthesis-depleted mutants are sensitive to soil-drying due to enhanced water loss. Our data demonstrate that sulfate is incorporated into Cys and tunes ABA biosynthesis in leaves, promoting stomatal closure, and that this mechanism contributes to the physiological water limitation response., (© 2018 American Society of Plant Biologists. All rights reserved.)

Phosphorus (P) constitutes one of five macronutrients essential for plant growth and development due to the central function of phosphate in energy metabolism, inheritance and metabolic control. In many ecosystems, plant available soil-P gets limited by soil aging. Hence, plants have developed adaptation strategies to cope with such limitation by an efficient plant and ecosystem internal P-cycling during annual growth. The natural floodplain habitat of fast-growing Populus × canescens is characterized by high soil-P availability. It was thus expected that the P-nutrition of P. × canescens had adapted to this conditions. Therefore, different P-fractions in different twig tissues were investigated during two annual growth cycles. The P-nutrition of P. × canescens markedly differs from that of European beech grown at low soil-P availability (Netzer F, Schmid C, Herschbach C, Rennenberg H (2017) Phosphorus-nutrition of European beech (Fagus sylvatica L.) during annual growth depends on tree age and P-availability in the soil. Environ Exp Bot 137:194-207). This was mainly due to a lack of tree internal P-cycling during annual growth indicated by the absence of P-storage and remobilization in twig bark and wood. Hence, strategies to economize P-nutrition and to prevent P-losses had not developed. This fits with the fast-growth strategy of P. × canescens at unrestricted P-availability. Hence, the P-nutrition strategy of P. × canescens can be seen as an evolutionary adaptation to its natural growth habitat., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Douglas fir (Pseudotsuga menziesii) is a conifer species that stores large amounts of terpenoids, mainly monoterpenoids in resin ducts of various tissues. The effects of drought on stored leaf terpenoid concentrations in trees are scarcely studied and published data are partially controversial, since reduced, unaffected or elevated terpenoid contents due to drought have been reported. Even less is known on the effect of drought on root terpenoids. In the present work, we investigated the effect of reduced water availability on the terpenoid content in roots and needles of Douglas fir seedlings. Two contrasting Douglas fir provenances were studied: an interior provenance (var. glauca) with assumed higher drought resistance, and a coastal provenance (var. menziesii) with assumed lower drought resistance. We tested the hypothesis that both provenances show specific patterns of stored terpenoids and that the patterns will change in response to drought in both, needles and roots. We further expected stronger changes in the less drought tolerant coastal provenance. For this purpose, we performed an experiment under controlled conditions, in which the trees were exposed to moderate and severe drought stress. According to our expectations, the study revealed clear provenance-specific terpenoid patterns in needles. However, such patterns were not detected in the roots. Drought slightly increased the needle terpenoid contents of the coastal but not of the interior provenance. We also observed increased terpenoid abundance mainly in roots of the moderately stressed coastal provenance. Overall, from the observed provenance-specific reactions with increased terpenoid levels in trees of the coastal origin in response to drought, we conclude on functions of terpenoids for abiotic stress tolerance that might be fulfilled by other, constitutively expressed mechanisms in drought-adapted interior provenances., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Forests store the largest terrestrial pools of carbon (C), helping to stabilize the global climate system, yet are threatened by climate change (CC) and associated air pollution (AP, highlighting ozone (O3) and nitrogen oxides (NOx)). We adopt the perspective that CC-AP drivers and physiological impacts are universal, resulting in consistent stress responses of forest ecosystems across zonobiomes. Evidence supporting this viewpoint is presented from the literature on ecosystem gross/net primary productivity and water cycling. Responses to CC-AP are compared across evergreen/deciduous foliage types, discussing implications of nutrition and resource turnover at tree and ecosystem scales. The availability of data is extremely uneven across zonobiomes, yet unifying patterns of ecosystem response are discernable. Ecosystem warming results in trade-offs between respiration and biomass production, affecting high elevation forests more than in the lowland tropics and low-elevation temperate zone. Resilience to drought is modulated by tree size and species richness. Elevated O3 tends to counteract stimulation by elevated carbon dioxide (CO2). Biotic stress and genomic structure ultimately determine ecosystem responsiveness. Aggrading early- rather than mature late-successional communities respond to CO2 enhancement, whereas O3 affects North American and Eurasian tree species consistently under free-air fumigation. Insect herbivory is exacerbated by CC-AP in biome-specific ways. Rhizosphere responses reflect similar stand-level nutritional dynamics across zonobiomes, but are modulated by differences in tree-soil nutrient cycling between deciduous and evergreen systems, and natural versus anthropogenic nitrogen (N) oversupply. The hypothesis of consistency of forest responses to interacting CC-AP is supported by currently available data, establishing the precedent for a global network of long-term coordinated research sites across zonobiomes to simultaneously advance both bottom-up (e.g., mechanistic) and top-down (systems-level) understanding. This global, synthetic approach is needed because high biological plasticity and physiographic variation across individual ecosystems currently limit development of predictive models of forest responses to CC-AP. Integrated research on C and nutrient cycling, O3-vegetation interactions and water relations must target mechanisms' ecosystem responsiveness. Worldwide case studies must be subject to biostatistical exploration to elucidate overarching response patterns and synthesize the resulting empirical data through advanced modelling, in order to provide regionally coherent, yet globally integrated information in support of internationally coordinated decision-making and policy development., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

In forest ecosystems, species use different strategies to increase their competitive ability for nitrogen (N) acquisition. The acquisition of N by trees is regulated by tree internal and environmental factors including mycorrhizae. In this study, we investigated the N uptake strategies of three co-occurring tree species [European beech (Fagus sylvatica L.), sycamore maple (Acer pseudoplatanus L.) and Norway maple (Acer platanoides L.)] in the understorey of a beech-dominated, N-limited forest on calcareous soil over two consecutive seasons. For this purpose, we studied (15)N uptake capacity as well as the allocation to N pools in the fine roots. Our results show that European beech had a higher capacity for both inorganic and organic N acquisition throughout the whole growing season compared with sycamore maple and Norway maple. The higher capacity of N acquisition in beech indicates a better adaption of beech to the understorey conditions of beech forests compared with the seedlings of other tree competitors under N-limited conditions. Despite these differences, all three species preferred organic over inorganic N sources throughout the growing season and showed similar seasonal patterns of N acquisition with an increased N uptake capacity in summer. However, this pattern varied with N source and year indicating that other environmental factors not assessed in this study further influenced N acquisition by the seedlings of the three tree species., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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 Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

We combined transcriptomic and biochemical approaches to study rhizobial and plant sulfur (S) metabolism in nitrogen (N) fixing nodules (Fix(+)) of Lotus japonicus, as well as the link of S-metabolism to symbiotic nitrogen fixation and the effect of nodules on whole-plant S-partitioning and metabolism. Our data reveal that N-fixing nodules are thiol-rich organs. Their high adenosine 5'-phosphosulfate reductase activity and strong (35)S-flux into cysteine and its metabolites, in combination with the transcriptional upregulation of several rhizobial and plant genes involved in S-assimilation, highlight the function of nodules as an important site of S-assimilation. The higher thiol content observed in nonsymbiotic organs of N-fixing plants in comparison to uninoculated plants could not be attributed to local biosynthesis, indicating that nodules are an important source of reduced S for the plant, which triggers whole-plant reprogramming of S-metabolism. Enhanced thiol biosynthesis in nodules and their impact on the whole-plant S-economy are dampened in plants nodulated by Fix(-) mutant rhizobia, which in most respects metabolically resemble uninoculated plants, indicating a strong interdependency between N-fixation and S-assimilation., (© 2015 American Society of Plant Biologists. All rights reserved.)

Rhizospheric nitric oxide (NO) and carbon dioxide (CO2) are signalling compounds known to affect physiological processes in plants. Their joint influence on tree nitrogen (N) nutrition, however, is still unknown. Therefore, this study investigated, for the first time, the combined effect of rhizospheric NO and CO2 levels on N uptake and N pools in European beech (Fagus sylvatica L.) seedlings depending on N availability. For this purpose, roots of seedlings were exposed to one of the nine combinations (i.e., low, ambient, high NO plus CO2 concentration) at either low or high N availability. Our results indicate a significant effect of rhizospheric NO and/or CO2 concentration on organic and inorganic N uptake. However, this effect depends strongly on NO and CO2 concentration, N availability and N source. Similarly, allocation of N to different N pools in the fine roots of beech seedlings also shifted with varying rhizospheric gas concentrations and N availability., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Understanding the dynamics of physiological process in the systems biology era requires approaches at the genome, transcriptome, proteome, and metabolome levels. In this context, metabolite flux experiments have been used in mapping metabolite pathways and analysing metabolic control. In the present review, sulphur metabolism was taken to illustrate current challenges of metabolic flux analyses. At the cellular level, restrictions in metabolite flux analyses originate from incomplete knowledge of the compartmentation network of metabolic pathways. Transport of metabolites through membranes is usually not considered in flux experiments but may be involved in controlling the whole pathway. Hence, steady-state and snapshot readings need to be expanded to time-course studies in combination with compartment-specific metabolite analyses. Because of species-specific differences, differences between tissues, and stress-related responses, the quantitative significance of different sulphur sinks has to be elucidated; this requires the development of methods for whole-sulphur metabolome approaches. Different cell types can contribute to metabolite fluxes to different extents at the tissue and organ level. Cell type-specific analyses are needed to characterize these contributions. Based on such approaches, metabolite flux analyses can be expanded to the whole-plant level by considering long-distance transport and, thus, the interaction of roots and the shoot in metabolite fluxes. However, whole-plant studies need detailed empirical and mathematical modelling that have to be validated by experimental analyses., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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 [Formula: see text], 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., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

In contrast to animal cells, plants use nitrate as a major source of nitrogen. Following the uptake of nitrate, this major macronutrient is fed into the vasculature for long-distance transport. The Arabidopsis thaliana shoot expresses the anion channel SLOW ANION CHANNEL1 (SLAC1) and its homolog SLAC1 HOMOLOGOUS3 (SLAH3), which prefer nitrate as substrate but cannot exclude chloride ions. By contrast, we identified SLAH2 as a nitrate-specific channel that is impermeable for chloride. To understand the molecular basis for nitrate selection in the SLAH2 channel, SLAC1 and SLAH2 were modeled to the structure of HiTehA, a distantly related bacterial member. Structure-guided site-directed mutations converted SLAC1 into a SLAH2-like nitrate-specific anion channel and vice versa. Our findings indicate that two pore-occluding phenylalanines constrict the pore. The selectivity filter of SLAC/SLAH anion channels is determined by the polarity of pore-lining residues located on alpha helix 3. Changing the polar character of a single amino acid side chain (Ser-228) to a nonpolar residue turned the nitrate-selective SLAH2 into a chloride/nitrate-permeable anion channel. Thus, the molecular basis of the anion specificity of SLAC/SLAH anion channels seems to be determined by the presence and constellation of polar side chains that act in concert with the two pore-occluding phenylalanines., (© 2014 American Society of Plant Biologists. All rights reserved.)

Does Dionaea muscipula, the Venus flytrap, use a particular mechanism to attract animal prey? This question was raised by Charles Darwin 140 years ago, but it remains unanswered. This study tested the hypothesis that Dionaea releases volatile organic compounds (VOCs) to allure prey insects. For this purpose, olfactory choice bioassays were performed to elucidate if Dionaea attracts Drosophila melanogaster. The VOCs emitted by the plant were further analysed by GC-MS and proton transfer reaction-mass spectrometry (PTR-MS). The bioassays documented that Drosophila was strongly attracted by the carnivorous plant. Over 60 VOCs, including terpenes, benzenoids, and aliphatics, were emitted by Dionaea, predominantly in the light. This work further tested whether attraction of animal prey is affected by the nutritional status of the plant. For this purpose, Dionaea plants were fed with insect biomass to improve plant N status. However, although such feeding altered the VOC emission pattern by reducing terpene release, the attraction of Drosophila was not affected. From these results it is concluded that Dionaea attracts insects on the basis of food smell mimicry because the scent released has strong similarity to the bouquet of fruits and plant flowers. Such a volatile blend is emitted to attract insects searching for food to visit the deadly capture organ of the Venus flytrap.

Plant species use different strategies for maximizing growth and fitness under changing environmental conditions. At the ecosystem level, seedlings in particular compete with other vegetation components for light and nitrogen (N), which often constitute growth-limiting resources. In this study, we investigated the effect of light availability on the competition for N between seedlings of European beech and sycamore maple and analysed the consequences of this competition for the composition of N metabolites in fine roots. Our results show different strategies in N acquisition between beech and sycamore maple. Both species responded to reduced light availability by adapting their morphological and physiological traits with a decrease in biomass and net assimilation rate and an increase in specific leaf area and leaf area ratio. For beech seedlings, competition with sycamore maple led to a reduction in organic N uptake capacity. Reduced light availability led to a decrease in ammonium, but an increase in glutamine-N uptake capacity in sycamore maple. However, this response was stronger compared with that of beech and was accompanied by reduced growth. Thus, our results suggest better adaptation of N acquisition to reduced light availability in beech compared with sycamore maple seedlings.

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.

Climate change as projected for Central Europe will lead to prolonged periods of summer drought and enhanced air temperature. Thus, forest management practices are required to take into account how species performance is adapted to cope with these climate changes. Oak trees may play a major role in future forests because of their relative drought-tolerance compared with other species like beech. Therefore, this study investigated the stress responses (i.e., anti-oxidants, free amino acids) in the leaves of three widely distributed oak species in Central Europe (i.e., Quercus robur L., Q. petraea [Matt.] Libel., Q. pubescens Willd.) to drought, air warming and the combination of drought plus air warming under controlled conditions after periods of spring drought, a short rewetting and summer drought. We quantified foliar levels of thiols, ascorbate, and free amino compounds in Q robur, Q. petraea and Q. pubescens. Our study showed that oak saplings had increased levels of γ-glutamylcysteine and total glutathione and proline with drought and air warming. Foliar ascorbate, glutathione disulfide and dehydroascorbic acid levels were not affected. The comparison of stress responses to drought and/or air warming between the three species showed higher foliar thiol levels in Q. robur and Q. pubescens compared with Q. petraea. For total and reduced ascorbic acid and γ-aminobutyric acid, the highest levels were found in Q. robur. In conclusion, our study showed that foliar anti-oxidant and free amino acid levels were significantly affected by drought plus air warming; however, this effect was species-dependent with the drought-tolerant species of Q. pubescens having the highest reactive oxygen species scavenging capacity among three tested oak species. Furthermore, stress responses as shown by increased levels of foliar anti-oxidants and free amino acids differ between calcareous and acidic soil indicating that the capacities of anti-oxidative defense and osmotic stress adjustment developed better on calcareous compared with acidic soil; however, this effect was metabolite- as well as species-specific.

Symbiotic nitrogen fixation (SNF) involves global changes in gene expression and metabolite accumulation in both rhizobia and the host plant. In order to study the metabolic changes mediated by leaf-root interaction, photosynthesis was limited in leaves by exposure of plants to darkness, and subsequently gene expression was profiled by real-time reverse transcription-PCR (RT-PCR) and metabolite levels by gas chromatography-mass spectrometry in the nodules of the model legume Lotus japonicus. Photosynthetic carbon deficiency caused by prolonged darkness affected many metabolic processes in L. japonicus nodules. Most of the metabolic genes analysed were down-regulated during the extended dark period. In addition to that, the levels of most metabolites decreased or remained unaltered, although accumulation of amino acids was observed. Reduced glycolysis and carbon fixation resulted in lower organic acid levels, especially of malate, the primary source of carbon for bacteroid metabolism and SNF. The high amino acid concentrations together with a reduction in total protein concentration indicate possible protein degradation in nodules under these conditions. Interestingly, comparisons between amino acid and protein content in various organs indicated systemic changes in response to prolonged darkness between nodulated and non-nodulated plants, rendering the nodule a source organ for both C and N under these conditions.

The influence of sulphur (S) depletion on the expression of genes related to S metabolism, and on metabolite and plant hormone contents was analysed in young and mature leaves, fine roots, xylem sap, and phloem exudates of poplar (Populus tremula×Populus alba) with special focus on early consequences. S depletion was applied by a gradual decrease of sulphate availability. The observed changes were correlated with sulphate contents. Based on the decrease in sulphate contents, two phases of S depletion could be distinguished that were denominated as 'S limitation' and 'early S deficiency'. S limitation was characterized by improved sulphate uptake (enhanced root-specific sulphate transporter PtaSULTR1;2 expression) and reduction capacities (enhanced adenosine 5'-phosphosulphate (APS) reductase expression) and by enhanced remobilization of sulphate from the vacuole (enhanced putative vacuolar sulphate transporter PtaSULTR4;2 expression). During early S deficiency, whole plant distribution of S was impacted, as indicated by increasing expression of the phloem-localized sulphate transporter PtaSULTR1;1 and by decreasing glutathione contents in fine roots, young leaves, mature leaves, and phloem exudates. Furthermore, at 'early S deficiency', expression of microRNA395 (miR395), which targets transcripts of PtaATPS3/4 (ATP sulphurylase) for cleavage, increased. Changes in plant hormone contents were observed at 'early S deficiency' only. Thus, S depletion affects S and plant hormone metabolism of poplar during 'S limitation' and 'early S deficiency' in a time series of events. Despite these consequences, the impact of S depletion on growth of poplar plants appears to be less severe than in Brassicaceae such as Arabidopsis thaliana or Brassica sp.

Flooding is assumed to cause an energy crisis in plants because-due to a lack of O(2)-mitochondrial respiration is replaced by alcoholic fermentation which yields considerably less energy equivalents. In the present study, the effect of flooding on the carbon metabolism of flooding-tolerant pedunculate oak (Quercus robur L.) and flooding-sensitive European beech (Fagus sylvatica L.) seedlings was characterized. Whereas soluble carbohydrate concentrations dropped in roots of F. sylvatica, they were constant in Q. robur during flooding. At the same time, root alcohol dehydrogenase activities were decreased in beech but not in oak, suggesting substrate limitation of alcoholic fermentation in beech roots. Surprisingly, leaf and phloem sap sugar concentrations increased in both species but to a much higher degree in beech. This finding suggests that the phloem unloading process in flooding-sensitive beech was strongly impaired. It is assumed that root-derived ethanol is transported to the leaves via the transpiration stream. This mechanism is considered an adaptation to flooding because it helps avoid the accumulation of toxic ethanol in the roots and supports the whole plant's carbon metabolism by channelling ethanol into the oxidative metabolism of the leaves. A labelling experiment demonstrated that in the leaves of flooded trees, ethanol metabolism does not differ between flooded beech and oak, indicating that processes in the roots are crucial for the trees' flooding tolerance.

During symbiotic nitrogen fixation (SNF), the nodule becomes a strong sink for photosynthetic carbon. Here, it was studied whether nodule dark CO(2) fixation could participate in a mechanism for CO(2) recycling through C(4)-type photosynthesis. Differences in the natural δ(13)C abundance between Lotus japonicus inoculated or not with the N-fixing Mesorhizobium loti were assessed. (13)C labelling and gene expression of key enzymes of CO(2) metabolism were applied in plants inoculated with wild-type or mutant fix(-) (deficient in N fixation) strains of M. loti, and in non-inoculated plants. Compared with non-inoculated legumes, inoculated legumes had higher natural δ(13)C abundance and total C in their hypergeous organs and nodules. In stems, (13)C accumulation and expression of genes coding for enzymes of malate metabolism were greater in inoculated compared with non-inoculated plants. Malate-oxidizing activity was localized in stem xylem parenchyma, sieve tubes, and photosynthetic outer cortex parenchyma of inoculated plants. In stems of plants inoculated with fix(-) M. loti strains, (13)C accumulation remained high, while accumulation of transcripts coding for malic enzyme isoforms increased. A potential mechanism is proposed for reducing carbon losses during SNF by the direct reincorporation of CO(2) respired by nodules and the transport and metabolism of C-containing metabolites in hypergeous organs.

Beech seedlings originating from 11 German provenances with different climatic conditions were grown in pots and cultivated in a greenhouse. The composition of macro- and microelements in roots, axes and leaves was measured after half of the seedlings were subjected to a simulated summer drought. The recently described sensitivity of these provenances to drought was compared with drought-mediated changes in the elemental and ionic composition in organs of the seedlings; in addition, partitioning between roots and shoots was evaluated. A number of element concentrations were decreased in roots due to drought (K 94% of control, Mg 94%, Mn 75% and Zn 85%). However, chloride concentration increased in all organs (115-125%) and was the only element affected in leaves. Some changes in ionome can be related to sensitivity of provenances, but it is difficult to decide whether these changes are a result of, or a reason for, drought tolerance or sensitivity. Observed increases in chloride concentration in all plant parts of drought-treated beech seedlings can be explained by its function in charge balance, in particular since the level of phosphate was reduced. As a result of chloride accumulation, the sum of added charges of anions (and cations) in water extracts of leaf and root material was similar between drought and control plants. Since only the partitioning of Ca and Al (both only in axis) as well as Mn was affected and other elements (together with previously observed effects on C, N, S and P) remained unaffected by drought in all provenances, it can be concluded that direct effects by means of mass flow inhibition in xylem and phloem are unlikely. Secondary effects, for example on the pH of transport sap and the apoplastic space, cannot be excluded from the present study. These effects may affect partitioning between the apoplast and symplast and therefore may be significant for drought sensitivity.

To investigate the impact of organic N compounds for inorganic nitrogen uptake in the rhizosphere, we fed ammonium nitrate with or without amino acids (i.e., glutamine or arginine) to the roots of non-mycorrhizal beech (Fagus sylvatica L.) seedlings under controlled conditions at different levels of N availability. Uptake of individual N sources was determined from ¹⁵N (inorganic N) and ¹⁵N ¹³C (organic N) accumulation in the roots. In addition, gene fragments encoding proteins involved in N uptake and metabolism were cloned from beech for gene expression analyses by quantitative real-time PCR in the roots. Generally, ammonium was preferred over nitrate as N source. Organic N sources were taken up by beech roots as intact molecules. Uptake of organic N was significantly higher than inorganic N uptake, thus contributing significantly to N nutrition of beech. Depending on the level of N availability, inorganic N uptake was negatively affected by the presence of organic N sources. This result indicates an overestimation of the contribution of inorganic N uptake to N nutrition of beech in previous studies. Apparently, association with mycorrhizal fungi is not essential for organic N uptake by beech roots. Gene expression analyses showed that transcriptional regulation of the amino acid transporters FsCAT3, FsCAT5, FsAAT and FsAAP and the ammonium transporter FsAMT1.2 in the roots is involved in N nutrition of beech.

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.

Seasonal tree-internal nitrogen cycling is an important strategy for trees to achieve high efficiency in the use of nitrogen (N). Key processes of this N redistribution are autumnal leaf senescence and storage of released N as bark storage proteins (BSP) in perennial tissues. While the regulation of leaf senescence has been intensively analysed in trees, the coordination of the complementary storage processes is still poorly understood. Therefore, we ascertained relationships between physiological-level and molecular-level processes and environmental factors under natural conditions in the bark of Populus x canescens. We analysed amino-N concentrations, total soluble protein concentration and transcript abundances of BSP genes in the bark of field-grown P. x canescens harvested during two annual growth cycles. By correlation analysis and linear modelling, we assessed interactions between biological data and meteorological conditions. Day length correlated with BSP expression, and air temperature correlated strongly with total protein concentration (r = -0.92), gamma-aminobutyric acid (GABA; r = 0.76) and arginine (r = -0.70). GABA and arginine also correlated significantly with total protein concentration and transcript abundances of BSP genes. We conclude that GABA and arginine potentially contribute to adjust storage processes in the bark of poplar trees to seasonal changes in environmental conditions.

Nut yield is highly variable in commercial macadamia production, and to ensure that nitrogen (N) supply does not limit yield, high rates of N fertilizer are generally applied. To elucidate N source and sink relations in mature Macadamia integrifolia Maiden et Betche trees, we traced (15)N label after injection into individual branches and, after soil application, analysed xylem sap and examined the effects of hedging on tree N relations. Xylem sap N and sugar composition and concentration changed in relation to phenology and tree management. Canopy position did not affect xylem sap N concentration but sampling date had a significant effect. Hedging in spring was associated with a rapid and dramatic reduction of the concentration of xylem sap N until the following autumn, but unhedged trees were not available to unequivocally assess the significance of the results. Following (15)N-branch injection in winter, most (15)N label was incorporated into flushing leaves and into bark. After (15)N injection in spring, flushing leaves and flowers were most strongly (15)N-labelled. In late spring, (15)N label was equally incorporated by developing nuts that were retained or later abscised. Soil (15)N application in summer resulted in (15)N-labelling of outer and mid-canopy leaves. In the following spring, (15)N label was translocated to flushing leaves, flowers and developing nuts. The results indicate that outer and mid-canopy leaves are the main N sink for soil-derived N during the vegetative phase and a N source for developing tissues during the reproductive phase. Our study provides evidence that N supply to developing nuts is not a primary cause for nut abscission, supporting the notion that high N fertilizer application rates do not improve nut retention. We propose that current orchard design and hedging practices should be reviewed in context of the role of outer canopy leaves as a source of N for reproductive tissues.

Glutathione (GSH) and ascorbate (ASC) are important antioxidants that are involved in stress defence and cell proliferation of meristematic root cells. In principle, synthesis of ASC and GSH in the roots as well as ASC and GSH transport from the shoot to the roots by phloem mass flow is possible. However, it is not yet known whether the ASC and/or the GSH level in roots depends on the supply from the shoot. This was analysed by feeding mature leaves with [(14)C]ASC or [(35)S]GSH and subsequent detection of the radiolabel in different root fractions. Quantitative dependency of root ASC and GSH on shoot-derived ASC and GSH was investigated with poplar (Populus tremula X P. alba) trees interrupted in phloem transport. [(35)S]GSH is transported from mature leaves to the root tips, but is withdrawn from the phloem along the entire transport path. When phloem transport was interrupted, the GSH content in root tips halved within 3 d. [(14)C]ASC is also transported from mature leaves to the root tips but, in contrast to GSH, ASC is not removed from the phloem along the transport path. Accordingly, ASC accumulates in root tips. Interruption of phloem transport disturbed the level and the ASC redox state within the entire root system. Diminished total ASC levels were attributed mainly to a decline of dehydroascorbate (DHA). As the redox state of ASC is of particular significance for root growth and development, it is concluded that phloem transport of ASC may constitute a shoot to root signal to coordinate growth and development at the whole plant level.

Sulphate assimilation provides reduced sulphur for the synthesis of cysteine, methionine, and numerous other essential metabolites and secondary compounds. The key step in the pathway is the reduction of activated sulphate, adenosine 5'-phosphosulphate (APS), to sulphite catalysed by APS reductase (APR). In the present study, [(35)S]sulphur flux from external sulphate into glutathione (GSH) and proteins was analysed to check whether APR controls the flux through the sulphate assimilation pathway in poplar roots under some stress conditions and in transgenic poplars. (i) O-Acetylserine (OAS) induced APR activity and the sulphur flux into GSH. (ii) The herbicide Acetochlor induced APR activity and results in a decline of GSH. Thereby the sulphur flux into GSH or protein remained unaffected. (iii) Cd treatment increased APR activity without any changes in sulphur flux but lowered sulphate uptake. Several transgenic poplar plants that were manipulated in sulphur metabolism were also analysed. (i) Transgenic poplar plants that overexpressed the gamma-glutamylcysteine synthetase (gamma-ECS) gene, the enzyme catalysing the key step in GSH formation, showed an increase in sulphur flux into GSH and sulphate uptake when gamma-ECS was targeted to the cytosol, while no changes in sulphur flux were observed when gamma-ECS was targeted to plastids. (ii) No effect on sulphur flux was observed when the sulphite oxidase (SO) gene from Arabidopsis thaliana, which catalyses the back reaction of APR, that is the reaction from sulphite to sulphate, was overexpressed. (iii) When Lemna minor APR was overexpressed in poplar, APR activity increased as expected, but no changes in sulphur flux were observed. For all of these experiments the flux control coefficient for APR was calculated. APR as a controlling step in sulphate assimilation seems obvious under OAS treatment, in gamma-ECS and SO overexpressing poplars. A possible loss of control under certain conditions, that is Cd treatment, Acetochlor treatment, and in APR overexpressing poplar, is discussed.

In this study, the effects of different light intensities either in direct sunlight or in the shade crown of adult beech (Fagus sylvatica L.) trees on delta13C and Delta18O were determined under ambient (1 x O3) and twice-ambient (2 x O3) atmospheric ozone concentrations during two consecutive years (2003 and 2004). We analysed the isotopic composition in leaf bulk, leaf cellulose, phloem and xylem material and related the results to (a) meteorological data (air temperature, T and relative humidity, RH), (b) leaf gas exchange measurements (stomatal conductance, g(s); transpiration rate, E; and maximum photosynthetic activity, A(max)) and (c) the outcome of a steady-state evaporative enrichment model. Delta13C was significantly lower in the shade than in the sun crown in all plant materials, whilst Delta18O was increased significantly in the shade than in the sun crown in bulk material and cellulose. Elevated ozone had no effect on delta13C, although Delta18O was influenced by ozone to varied degrees during single months. We observed significant seasonal changes for both parameters, especially in 2004, and also significant differences between the study years. Relating the findings to meteorological data and gas exchange parameters, we conclude that the differences in Delta18O between the sun and the shade crown were predominantly caused by the Péclet effect. This assumption was supported by the modelled Delta18O values for leaf cellulose. It was demonstrated that independent of RH, light-dependent reduction of stomatal conductance (and thus transpiration) and of A(max) can drive the pattern of Delta18O increase with the concomitant decrease of delta13C in the shade crown. The effect of doubling ozone levels on time-integrated stomatal conductance and transpiration as indicated by the combined analysis of Delta18O and delta13C was much lower than the influence caused by the light exposure.

Isoprene is the most abundant volatile compound emitted by vegetation. It influences air chemistry and is thought to take part in plant defense reactions against abiotic stress such as high temperature or ozone. However, whether or not isoprene emission impacts ozone tolerance of plants is still in discussion. In this study, we exploited the transgenic non-isoprene emitting grey poplar (Populus x canescens (Aiton) Sm.) in a biochemical and physiological model study to investigate the effect of acute ozone stress on the elicitation of defense-related emissions of plant volatiles, on photosynthesis and on the antioxidative system. We recorded that non-isoprene emitting poplars were more resistant to ozone as indicated by less damaged leaf area and higher assimilation rates compared to ozone-exposed wild-type (WT) plants. The integral of green leaf volatile emissions was different between the two poplar phenotypes and was a reliable early marker for subsequent leaf damage. For other stress-induced volatiles, such as mono-, homo- and sesquiterpenes and methyl salicylate, similar time profiles, pattern and emission intensities were observed in both transgenic and WT plants. However, unstressed non-isoprene emitting poplars are characterized by elevated levels of ascorbate and alpha-tocopherol as well as by a more effective de-epoxidation ratio of xanthophylls than the WT. Since ozone quenching properties of ascorbate are much higher than those of isoprene and furthermore alpha-tocopherol is also an essential antioxidant, non-isoprene emitting poplars might benefit from changes within the antioxidative system by providing them with enhanced ozone tolerance.

In southeastern Australia, the overstory species Eucalyptus regnans F. Muell. commonly grows with either of the two leguminous understory trees, Acacia melanoxylon (R. Br. Ex Ait. f.) or Acacia dealbata (Link.). Our objective was to elucidate interactions between the dominant eucalypt and its companion acacias for nitrogen (N) sources. Use of stable N isotopes as tracers revealed that ammonium was the preferred soil N source for all species, nevertheless, total N uptake varied greatly among species. Studies with double-labeled ((13)C/(15)N) glutamine indicated the uptake of this form of organic N in small amounts by both E. regnans and the Acacia spp. These and other data imply that, in contrast to boreal forests, organic N is not a significant component of N nutrition in mountain ash forests. Field and laboratory studies provided evidence that N(2)-fixation capacity of acacias varies with stand development, with N-fixing species playing an important role in N nutrition during the early but not the mature stages of forest growth. An index of N-uptake efficiency - the amount of oxygen consumed per unit N taken up - was compared across four N sources and three species. Nitrate uptake was the least efficient form of N acquisition, especially compared with ammonium uptake which was up to 30-fold less costly. Efficiency of glutamine uptake was intermediate between that of ammonium and nitrate. Differences in uptake efficiency among N forms were most pronounced for the Acacia spp. and least for E. regnans. We conclude that an overlap in requirements among sympatric Acacia spp. and E. regnans for specific soil N sources can be bypassed because of changes in biochemical strategies of Acacia spp. triggered by increasing soil N concentrations during stand development. Further studies might elucidate whether this is a common feature of complex forest ecosystems, or a specialty of the interaction between eucalypts and acacias.

The waterlogging tolerance and the physiological responses to this stress were tested in seedlings of Fraxinus angustifolia, an ash tree inhabiting riparian forests, and two provenances of the closely related Fraxinus excelsior, one derived from a riparian forest (FER) and one from a mountainous region (FEM). Besides visible damage, physiological parameters reflecting adaptations of plants to waterlogging such as net CO(2) assimilation, alcoholic fermentation, and the concentrations of metabolites related to flooding responses were studied. Consistent with the higher flooding tolerance of F. angustifolia and FER compared with FEM, net assimilation remained unaffected in F. angustifolia, was slightly reduced in FER, but was strongly affected in FEM. Altered carbohydrate concentrations in the roots of the seedlings suggest differences in the ability to supply alcoholic fermentation with substrate during prolonged periods of soil anoxia. Another difference between the seedlings was connected to the gamma-aminobutyric acid (GABA) shunt which resulted in alanine accumulation in the flooding-tolerant trees, but strong GABA accumulation in the more sensitive FEM seedlings. This finding indicates differences in GABA conversion into alanine which might result in an accumulation of phytotoxic levels of intermediates. Such provenance-specific differences in Common ash suggest that the selection of appropriate provenances is essential for forest management in flood-prone areas.

We quantified ascorbate, glutathione and alpha-tocopherol in fine roots of mature Fagus sylvatica L. under free-air canopy ozone (O(3)) exposure (twice ambient O(3) concentration, 2x[O(3)]) during two growing seasons that differed in the extent of summer drought (exceptional drought year 2003, average year 2004). This design allowed us to test whether O(3) exposure or drought, or both, affected root antioxidants during the growing season. In both years, root ascorbate and alpha-tocopherol showed a similar relationship with volumetric soil water content (SWC): ascorbate concentrations on a root dry mass basis increased from about 6 to 12 micromol g(-1) when SWC dropped from 25 to 20%, and a-tocopherol increased from 100 to 150 nmol g(-1) at SWC values below 20%. Root glutathione showed no relationship with SWC or differences between the dry and the average year, but it was significantly and consistently diminished by 2x[O(3)]. Our results were inconclusive as to whether shoot-root translocation of glutathione or glutathione production in the roots was diminished. Phloem glutathione concentrations in the canopy remained constant, but reduced transport velocity in the phloem and, as a consequence, reduced mass flow of glutathione cannot be ruled out.

Pedospheric nitrate uptake is closely integrated with the nitrogen (N) status and demand of the whole tree. Signaling substances communicating the N demand of the shoot to the roots are required in an integrated regulatory system. Besides phloem mobility, such signal compounds must have the potential to repress or increase nitrate uptake either at the transcriptional or post-transcriptional level. Amino compounds cycling within the tree are involved in the regulation of nitrate uptake. In many tree species, inorganic N is generally assimilated in roots, and amino acids--the direct products of N assimilation--are transported in the xylem to the sites of N demand. If the quantity of amino acids transported to the above-ground parts of the tree exceeds shoot N demand, some amino compounds are reallocated to the roots by phloem transport. Particular amino compounds exert transcriptional and post-transcriptional control over nitrate uptake by roots. Induction of nitrate transporters is mediated by nitrate or nitrite, or both, and possibly also by cytokinins, which cycle within the tree and act as both root-to-shoot and shoot-to-root signals. This review focuses on tree-specific requirements for N regulation and signaling, as well as the link between carbon metabolism and nitrate uptake.

Elevated concentrations of carbon dioxide ([CO2]) and ozone ([O3]) affect primary metabolism of trees in opposite ways. We studied their potential interactions on carbohydrate concentrations and contents. Two hypotheses currently under debate were tested. (1) Stimulation of primary metabolism by prolonged exposure to elevated [CO2] does not compensate for the adverse effects of O3 on carbohydrate accumulation and biomass partitioning to the root. (2) Growth in a mixed-species planting will repress plant responses to elevated [O3] and [CO2] relative to conditions in a monoculture. To this end, European beech (Fagus sylvatica L.) and Norway spruce (Picea abies (L.) Karst.) saplings grown under conditions of intra- and interspecific competition were pre-acclimated for 1 year to ambient or elevated [CO2]. In the following 2-year phytotron study, trees were exposed to factorial combinations of ambient and elevated [O3] and [CO2]. The total carbohydrate content (sugar and starch) of spruce was greater in plants exposed to elevated [CO2] than in plants exposed to ambient [CO2]. In beech, the opposite response was observed, especially when this species was grown in combination with spruce. Overall, the data did not support Hypothesis 1, because the adverse effects of O3 were counteracted by elevated [CO2]. Support for Hypothesis 2 was species-dependent. In beech saplings, reduction of carbohydrates by elevated [O3] and stimulation by elevated [CO2] were repressed by competitive interaction with spruce. In contrast, in spruce, stimulation of carbohydrates by elevated [CO2] was similar in mono- and mixed cultures. Thus Hypothesis 2 was supported for beech but not spruce. We conclude that, in juvenile beech and spruce, a 3-year exposure to elevated [CO2] counteracts the adverse effects of O3 on carbohydrate concentrations and contents. For beech, sensitivity to elevated [CO2] and [O3] was high in monoculture but was largely repressed by interspecific competition with spruce. In contrast, the response of spruce to perturbations of atmospheric chemistry was not significantly affected by either intra- or interspecific competition.

Sulphate assimilation is an essential pathway being a source of reduced sulphur for various cellular processes and for the synthesis of glutathione, a major factor in plant stress defence. Many reports have shown that sulphate assimilation is well co-ordinated with the assimilation of nitrate and carbon. It has long been known that, during nitrate deficiency, sulphate assimilation is reduced and that the capacity to reduce nitrate is diminished in plants starved for sulphate. Only recently, however, was it shown that adenosine 5' phosphosulphate reductase (APR), the key enzyme of sulphate assimilation, is regulated by carbohydrates. In plants treated with sucrose or glucose APR was induced, whereas the activity was strongly reduced in plants grown in CO(2)-free air. The availability of cysteine is a crucial factor in glutathione synthesis, but an adequate supply of glutamate and glycine are also important. The molecular mechanisms for the co-ordination of S, N, and C assimilation are not known. O-acetylserine, a precursor of cysteine, was proposed to be the signal regulating sulphate assimilation, but most probably is not the outgoing signal to N and C metabolism. cDNA arrays revealed the induction of genes involved in auxin synthesis upon S-starvation, pointing to a possible role of phytohormones. Clearly, despite significant progress in understanding the regulation of sulphate assimilation and glutathione synthesis, their co-ordination with N and C metabolism achieved, and several potential signal molecules identified, present knowledge is still far from being sufficient.

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.

Glutathione (GSH) is the major low molecular weight thiol in plants with different functions in stress defence and the transport and storage of sulphur. Its synthesis is dependent on the supply of its constituent amino acids cysteine, glutamate, and glycine. GSH is a feedback inhibitor of the sulphate assimilation pathway, the primary source of cysteine synthesis. Sulphate assimilation has been analysed in transgenic poplars (Populus tremula x P. alba) overexpressing gamma-glutamylcysteine synthetase, the key enzyme of GSH synthesis, and the results compared with the effects of exogenously added GSH. Although foliar GSH levels were 3-4-fold increased in the transgenic plants, the activities of enzymes of sulphate assimilation, namely ATP sulphurylase, adenosine 5'-phosphosulphate reductase (APR), sulphite reductase, serine acetyltransferase, and O-acetylserine (thiol)lyase were not affected in three transgenic lines compared with the wild type. Also the mRNA levels of these enzymes were not altered by the increased GSH levels. By contrast, an increase in GSH content due to exogenously supplied GSH resulted in a strong reduction in APR activity and mRNA accumulation. This feedback regulation was reverted by simultaneous addition of O-acetylserine (OAS). However, OAS measurements revealed that OAS cannot be the only signal responsible for the lack of feedback regulation of APR by GSH in the transgenic poplars.

With the aim of analysing the relative importance of sugar supply and nitrogen nutrition for the regulation of sulphate assimilation, the regulation of adenosine 5'-phosphosulphate reductase (APR), a key enzyme of sulphate reduction in plants, was studied. Glucose feeding experiments with Arabidopsis thaliana cultivated with and without a nitrogen source were performed. After a 38 h dark period, APR mRNA, protein, and enzymatic activity levels decreased dramatically in roots. The addition of 0.5% (w/v) glucose to the culture medium resulted in an increase of APR levels in roots (mRNA, protein and activity), comparable to those of plants kept under normal light conditions. Treatment of roots with d-sorbitol or d-mannitol did not increase APR activity, indicating that osmotic stress was not involved in APR regulation. The addition of O-acetyl-l-serine (OAS) also quickly and transiently increased APR levels (mRNA, protein, and activity). Feeding plants with a combination of glucose and OAS resulted in a more than additive induction of APR activity. Contrary to nitrate reductase, APR was also increased by glucose in N-deficient plants, indicating that this effect was independent of nitrate assimilation. [35S]-sulphate feeding experiments showed that the addition of glucose to dark-treated roots resulted in an increased incorporation of [35S] into thiols and proteins, which corresponded to the increased levels of APR activity. Under N-deficient conditions, glucose also increased thiol labelling, but did not increase the incorporation of label into proteins. These results demonstrate that (i) exogenously supplied glucose can replace the function of photoassimilates in roots; (ii) APR is subject to co-ordinated metabolic control by carbon metabolism; (iii) positive sugar signalling overrides negative signalling from nitrate assimilation in APR regulation. Furthermore, signals originating from nitrogen and carbon metabolism regulate APR synergistically.

14C-Gln, (14)C-Asp, (15)N-Gln, and (15)N-Asp were fed to cut tips of 2- or 3-year-old needles of spruce twigs, still attached to the tree. After incubation, distribution of the radiolabel and (15)N enrichment was studied in needles, bark and wood tissues of girdled twigs and untreated controls. Analysis of the twig tissues showed that between 22% and 26% of the total amount of the tracers applied had been taken up. Since export out of the application segment and distribution between needles, bark and wood was comparable for (14)C and (15)N tracer, it was concluded that, mainly the amino compounds that had been fed were subject to long- distance transport within the plant and supplied the new sprout with nitrogen. Asp was exported to a greater extent to developing needles compared with Gln. This difference in export between the two amino compounds applied may be explained by the different pool sizes of Gln and Asp in xylem and phloem or differences in xylem and phloem loading. Girdling of the stem showed that the transport of reduced nitrogen compounds from older needle generations to current-year needles proceeded in both xylem and phloem. In addition, an intensive bidirectional exchange of Gln and Asp between xylem and phloem was observed during long-distance transport.