The association of protein-bound methionine sulfoxide with proteomic basis for aging in beech seeds.

BACKGROUND: European beech (Fagus sylvatica L.) trees produce seeds irregularly; therefore, it is necessary to store beech seeds for forestation. Despite the acquisition of desiccation tolerance during development, beech seeds are classified as intermediate because they lose viability during long-term storage faster than typical orthodox seeds. In this study, beech seeds stored for short (3 years) or long (20 years) periods under optimal conditions and displaying 92 and 30% germination capacity, respectively, were compared. RESULTS: Aged seeds displayed increased membrane damage, manifested as electrolyte leakage and lipid peroxidation levels. Analyses have been based on embryonic axes, which contained higher levels of reactive oxygen species (ROS) and higher levels of protein-bound methionine sulfoxide (MetO) in aged seeds. Using label-free quantitative proteomics, 3,949 proteins were identified, of which 2,442 were reliably quantified pointing to 24 more abundant proteins and 35 less abundant proteins in beech seeds under long-term storage conditions. Functional analyses based on gene ontology annotations revealed that nucleic acid binding activity (molecular function), ribosome organization or biogenesis and transmembrane transport (cellular processes), translational proteins (protein class) and membranous anatomical entities (cellular compartment) were affected in aged seeds. To verify whether MetO, the oxidative posttranslational modification of proteins that can be reversed via the action of methionine sulfoxide reductase (Msr) enzymes, is involved in the aging of beech seeds, we identified and quantified 226 MetO-containing proteins, among which 9 and 19 exhibited significantly up- and downregulated MetO levels, respectively, in beech seeds under long-term storage conditions. Several Msr isoforms were identified and recognized as MsrA1-like, MsrA4, MsrB5 and MsrB5-like in beech seeds. Only MsrA1-like displayed decreased abundance in aged seeds. CONCLUSIONS: We demonstrated that the loss of membrane integrity reflected in the elevated abundance of membrane proteins had a higher impact on seed aging progress than the MetO/Msr system. Proteome analyses enabled us to propose protein Sec61 and glyceraldehyde-3-phosphate dehydrogenase as potential longevity modulators in beech seeds.

Long-term behaviour of Cs-137, Cs-133 and K in beech trees of French forests.

In the long-term after atmospheric deposit onto a forest ecosystem, Cs-137 becomes incorporated into the biogeochemical cycle of stable elements and progressively reaches a quasi-equilibrium state. This study aimed at determining to what extent Cs-137 activity distribution in tree vegetation could be predicted from that of stable caesium (Cs-133) and potassium (K), which are known to be stable chemical analogues and competitors for Cs-137 intake in tree organs. Field campaigns that focused on beech trees (Fagus sylvatica L.) were conducted in 2021 in three French forest stands with contrasted characteristics regarding either the contribution of global vs. Chornobyl fallouts, soil or climatic conditions. Decades after Cs-137 fallouts, it was found that more than 80% of the total radioactive inventory in the system remained confined in the top 20 cm mineral layers, while organic layers and beech vegetation (including roots) contributed each to less than 1.5%. The enhanced downward migration of Cs-137 in cambisol than podzol forest sites was presumably due to migration of clay particles and bioturbation. The distribution of Cs-137 and Cs-133 inventories in beech trees was very similar among sites but differed from that of K due a higher accumulation of Cs isotopes in roots (40-50% vs. < 25% for K). The aggregated transfer factor (Tag) of Cs-137 calculated for aerial beech organs were all lower than those reported in literature more than 20 years ago, this suggesting a decrease of bioavailability in soil due to ageing processes. Regarding their variability, Tags were generally lower by a factor 5 at the cambisol site, which was fairly well explained by a much higher value of RIP (radiocesium immobilisation potential). Cs-137 concentrations in trees organs normalized by the soil exchangeable fractions were linearly correlated to those of Cs-133 and the best fit was found for the linear regression model without intercept indicating that no more contribution of the foliar uptake could be observed on long term. Provided that the vertical distribution of caesium concentrations and fine root density are properly measured or estimated, Cs-133 was shown to be a much better proxy than K to estimate the root transfer of Cs-137.

Substantial uptake of nitrous oxide (N2O) by shoots of mature European beech.

Similar to soils, tree stems emit and consume nitrous oxide (N2O) from the atmosphere. Although tree leaves dominate tree surface area, they have been completely excluded from field N2O flux measurements and therefore their role in forest N2O exchange remains unknown. We explored the contribution of leaf fluxes to forest N2O exchange. We determined the N2O exchange of mature European beech (Fagus sylvatica) stems and shoots (i.e., terminal branches) and of adjacent forest floor, in a typical temperate upland forest in Germany. The beech stems, and particularly the shoots, acted as net N2O sinks (-0.254 ± 0.827 µg N2O m-2 stem area h-1 and -4.54 ± 1.53 µg N2O m-2 leaf area h-1, respectively), while the forest floor was a net source (2.41 ± 1.08 µg N2O m-2 soil area h-1). The unstudied tree shoots were identified as a significant contributor to the net ecosystem N2O exchange. Moreover, we revealed for the first time that tree leaves act as substantial N2O sinks. Although this is the first study of its kind, it is of global importance for the proper design of future flux studies in forest ecosystems worldwide. Our results demonstrate that excluding tree leaves from forest N2O flux measurements can lead to misinterpretation of tree and forest N2O exchange, and thus global forest greenhouse gas flux inventories.

Does long-term drought or repeated defoliation affect seasonal leaf N cycling in young beech trees?

Forest trees adopt effective strategies to optimize nitrogen (N) use through internal N recycling. In the context of more recurrent environmental stresses due to climate change, the question remains of whether increased frequency of drought or defoliation threatens this internal N recycling strategy. We submitted 8-year-old beech trees to 2 years of either severe drought (Dro) or manual defoliation (Def) to create a state of N starvation. At the end of the second year before leaf senescence, we labeled the foliage of the Dro and Def trees, as well as that of control (Co) trees, with 15N-urea. Leaf N resorption, winter tree N storage (total N, 15N, amino acids, soluble proteins) and N remobilization in spring were evaluated for the three treatments. Defoliation and drought did not significantly impact foliar N resorption or N concentrations in organs in winter. Total N amounts in Def tree remained close to those in Co tree, but winter N was stored more in the branches than in the trunk and roots. Total N amount in Dro trees was drastically reduced (-55%), especially at the trunk level, but soluble protein concentrations increased in the trunk and fine roots compared with Co trees. During spring, 15N was mobilized from the trunk, branches and twigs of both Co and Def trees to support leaf growth. It was only provided through twig 15N remobilization in the Dro trees, thus resulting in extremely reduced Dro leaf N amounts. Our results suggest that stress-induced changes occur in N metabolism but with varying severity depending on the constraints: within-tree 15N transport and storage strategy changed in response to defoliation, whereas a soil water deficit induced a drastic reduction of the N amounts in all the tree organs. Consequently, N dysfunction could be involved in drought-induced beech tree mortality under the future climate.

A multifaceted approach to reveal the very-fine root's response of Fagus sylvatica seedlings to different drought intensities.

How temperate trees respond to drier summers strongly depends on the drought susceptibility and the starch reserve of the very-fine roots (<0.5 mm in diameter). We performed morphological, physiological, chemical, and proteomic analyses on very-fine roots of Fagus sylvatica seedlings grown under moderate- and severe drought conditions. Moreover, to reveal the role of the starch reserves, a girdling approach was adopted to interrupt the flux of photosynthates toward the downstream sinks. Results show a seasonal sigmoidal growth pattern without evident mortality under moderate drought. After the severe-drought period, intact plants showed lower starch concentration and higher growth than those subjected to moderate drought, highlighting that very-fine roots rely on their starch reserves to resume growth. This behavior caused them to die with the onset of autumn, which was not observed under moderate drought. These findings indicated that extreme dry soil conditions are needed for significant root death in beech seedlings and that mortality mechanisms are defined within individual compartments. The girdling treatment showed that the physiological responses of very-fine roots to severe drought stress are critically related to the altered load or the reduced transport velocity of the phloem and that the changes in starch allocation critically alter the distribution of biomass. Proteomic evidence revealed that the phloem flux-dependent response was characterized by the decrease of carbon enzymes and the establishment of mechanisms to avoid the reduction of the osmotic potential. The response independent from the aboveground mainly involved the alteration of primary metabolic processes and cell wall-related enzymes.

First evidence of nanoparticle uptake through leaves and roots in beech (Fagus sylvatica L.) and pine (Pinus sylvestris L.).

Trees have been used for phytoremediation and as biomonitors of air pollution. However, the mechanisms by which trees mitigate nanoparticle pollution in the environment are still unclear. We investigated whether two important tree species, European beech (Fagus sylvatica L.) and Scots pine (Pinus sylvestris L.), are able to take up and transport differently charged gold nanoparticles (Au-NPs) into their stem by comparing leaf-to-root and root-to-leaf pathways. Au-NPs were taken up by roots and leaves, and a small fraction was transported to the stem in both species. Au-NPs were transported from leaves to roots but not vice versa. Leaf Au uptake was higher in beech than in pine, probably because of the higher stomatal density and wood characteristics of beech. Confocal (3D) analysis confirmed the presence of Au-NPs in trichomes and leaf blade, about 20-30 µm below the leaf surface in beech. Most Au-NPs likely penetrated into the stomatal openings through diffusion of Au-NPs as suggested by the 3D XRF scanning analysis. However, trichomes were probably involved in the uptake and internal immobilization of NPs, besides their ability to retain them on the leaf surface. The surface charge of Au-NPs may have played a role in their adhesion and uptake, but not in their transport to different tree compartments. Stomatal conductance did not influence the uptake of Au-NPs. This is the first study that shows nanoparticle uptake and transport in beech and pine, contributing to a better understanding of the interactions of NPs with different tree species.

Sugar concentrations and expression of SUTs suggest active phloem loading in tall trees of Fagus sylvatica and Quercus robur.

Phloem loading and sugar distribution are key steps for carbon partitioning in herbaceous and woody species. Although the phloem loading mechanisms in herbs are well studied, less is known for trees. It was shown for saplings of Fagus sylvatica L. and Quercus robur L. that the sucrose concentration in the phloem sap was higher than in the mesophyll cells, which suggests that phloem loading of sucrose involves active steps. However, the question remains whether this also applies for tall trees. To approach this question, tissue-specific sugar and starch contents of small and tall trees of F. sylvatica and Q. robur as well as the sugar concentration in the subcellular compartments of mesophyll cells were examined. Moreover, sucrose uptake transporters (SUTs) were analyzed by heterology expression in yeast and the tissue-specific expressions of SUTs were investigated. Sugar content in leaves of the canopy (11 and 26 m height) was up to 25% higher compared with that of leaves of small trees of F. sylvatica and Q. robur (2 m height). The sucrose concentration in the cytosol of mesophyll cells from tall trees was between 120 and 240 mM and about 4- to 8-fold lower than the sucrose concentration in the phloem sap of saplings. The analyzed SUT sequences of both tree species cluster into three types, similar to SUTs from other plant species. Heterologous expression in yeast confirmed that all analyzed SUTs are functional sucrose transporters. Moreover, all SUTs were expressed in leaves, bark and wood of the canopy and the expression levels in small and tall trees were similar. The results show that the phloem loading in leaves of tall trees of F. sylvatica and Q. robur probably involves active steps, because there is an uphill concentration gradient for sucrose. SUTs may be involved in phloem loading.

Aerial and underground organs display specific metabolic strategies to cope with water stress under rising atmospheric CO2 in Fagus sylvatica L.

Beech is known to be a moderately drought-sensitive tree species, and future increases in atmospheric concentrations of CO2 ([CO2 ]) could influence its ecological interactions, also with changes at the metabolic level. The metabolome of leaves and roots of drought-stressed beech seedlings grown under two different [CO2 ] (400 (aCO2 ) and 800 (eCO2 ) ppm) was analyzed together with gas exchange parameters and water status. Water stress estimated from predawn leaf water potential (Ψpd ) was similar under both [CO2 ], although eCO2 had a positive impact on net photosynthesis and intrinsic water use efficiency. The aerial and underground organs showed different metabolomes. Leaves mainly stored C metabolites, while those of N and P accumulated differentially in roots. Drought triggered the proline and N-rich amino acids biosynthesis in roots through the activation of arginine and proline pathways. Besides the TCA cycle, polyols and soluble sugar biosynthesis were activated in roots, with no clear pattern seen in the leaves, prioritizing the root functioning as metabolites sink. eCO2 slightly altered this metabolic acclimation to drought, reflecting mitigation of its effect. The leaves showed only minor changes, investing C surplus in secondary metabolites and malic acid. The TCA cycle metabolites and osmotically active substances increased in roots, but many other metabolites decreased as if the water stress was dampened. Above- and belowground plant metabolomes were differentially affected by two drivers of climate change, water scarcity and high [CO2 ], showing different chemical responsiveness that could modulate the tree adaptation to future climatic scenarios.

MR Study of Water Distribution in a Beech (Fagus sylvatica) Branch Using Relaxometry Methods.

Wood is a widely used material because it is environmentally sustainable, renewable and relatively inexpensive. Due to the hygroscopic nature of wood, its physical and mechanical properties as well as the susceptibility to fungal decay are strongly influenced by its moisture content, constantly changing in the course of everyday use. Therefore, the understanding of the water state (free or bound) and its distribution at different moisture contents is of great importance. In this study, changes of the water state and its distribution in a beech sample while drying from the green (fresh cut) to the absolutely dry state were monitored by 1D and 2D 1H NMR relaxometry as well as by spatial mapping of the relaxation times T1 and T2. The relaxometry results are consistent with the model of homogeneously emptying pores in the bioporous system with connected pores. This was also confirmed by the relaxation time mapping results which revealed the moisture transport in the course of drying from an axially oriented early- and latewood system to radial rays through which it evaporates from the branch. The results of this study confirmed that MRI is an efficient tool to study the pathways of water transport in wood in the course of drying and is capable of determining the state of water and its distribution in wood.

Localization and Dynamics of the Methionine Sulfoxide Reductases MsrB1 and MsrB2 in Beech Seeds.

Beech seeds are produced irregularly, and there is a need for long-term storage of these seeds for forest management practices. Accumulated reactive oxygen species broadly oxidize molecules, including amino acids, such as methionine, thereby contributing to decreased seed viability. Methionine oxidation can be reversed by the activity of methionine sulfoxide reductases (Msrs), which are enzymes involved in the regulation of many developmental processes and stress responses. Two types of Msrs, MsrB1 and MsrB2, were investigated in beech seeds to determine their abundance and localization. MsrB1 and MsrB2 were detected in the cortical cells and the outer area of the vascular cylinder of the embryonic axes as well as in the epidermis and parenchyma cells of cotyledons. The abundances of MsrB1 and MsrB2 decreased during long-term storage. Ultrastructural analyses have demonstrated the accumulation of these proteins in protein storage vacuoles and in the cytoplasm, especially in close proximity to the cell membrane. In silico predictions of possible Msr interactions supported our findings. In this study, we investigate the contribution of MsrB1 and MsrB2 locations in the regulation of seed viability and suggest that MsrB2 is linked with the longevity of beech seeds via association with proper utilization of storage material.

Comparative transcriptional analyses of Pleurotus ostreatus mutants on beech wood and rice straw shed light on substrate-biased gene regulation.

Distinct wood degraders occupying their preferred habitats have biased enzyme repertoires that are well fitted to their colonized substrates. Pleurotus ostreatus, commonly found on wood, has evolved its own enzyme-producing traits. In our previous study, transcriptional shifts in several P. ostreatus delignification-defective mutants, including Δhir1 and Δgat1 strains, were analyzed, which revealed the downregulation of ligninolytic genes and the upregulation of cellulolytic and xylanolytic genes when compared to their parental strain 20b on beech wood sawdust medium (BWS). In this study, rice straw (RS) was used as an alternative substrate to examine the transcriptional responses of P. ostreatus to distinct substrates. The vp1 gene and a cupredoxin-encoding gene were significantly upregulated in the 20b strain on RS compared with that on BWS, reflecting their distinct regulation patterns. The overall expression level of genes encoding glucuronidases was also higher on RS than on BWS, showing a good correlation with the substrate composition. Transcriptional alterations in the mutants (Δhir1 or Δgat1 versus 20b strain) on RS were similar to those on BWS, and the extracellular lignocellulose-degrading enzyme activities and lignin-degrading ability of the mutants on RS were consistent with the transcriptional alterations of the corresponding enzyme-encoding genes. However, transcripts of specific genes encoding enzymes belonging to the same CAZyme family exhibited distinct alteration patterns in the mutant strains grown on RS compared to those grown on BWS. These findings provide new insights into the molecular mechanisms underlying the transcriptional regulation of lignocellulolytic genes in P. ostreatus.Key Points• P. ostreatus expressed variable enzymatic repertoire-related genes in response to distinct substrates.• A demand to upregulate the cellulolytic genes seems to be present in ligninolysis-deficient mutants.• The regulation of some specific genes probably driven by the demand is dependent on the substrate.

Frost and drought: Effects of extreme weather events on stem carbon dynamics in a Mediterranean beech forest.

The effects of short-term extreme events on tree functioning and physiology are still rather elusive. European beech is one of the most sensitive species to late frost and water shortage. We investigated the intra-annual C dynamics in stems under such conditions. Wood formation and stem CO2 efflux were monitored in a Mediterranean beech forest for 3 years (2015-2017), including a late frost (2016) and a summer drought (2017). The late frost reduced radial growth and, consequently, the amount of carbon fixed in the stem biomass by 80%. Stem carbon dioxide efflux in 2016 was reduced by 25%, which can be attributed to the reduction of effluxes due to growth respiration. Counter to our expectations, we found no effects of the 2017 summer drought on radial growth and stem carbon efflux. The studied extreme weather events had various effects on tree growth. Even though late spring frost had a strong impact on beech radial growth in the current year, trees fully recovered in the following growing season, indicating high resilience of beech to this stressful event.

Identifying the pathways for foliar water uptake in beech (Fagus sylvatica L.): a major role for trichomes.

Foliar water uptake (FWU), the direct uptake of water into leaves, is a global phenomenon, having been observed in an increasing number of plant species. Despite the growing recognition of its functional relevance, our understanding of how FWU occurs and which foliar surface structures are implicated, is limited. In the present study, fluorescent and ionic tracers, as well as microcomputed tomography, were used to assess potential pathways for water entry in leaves of beech, a widely distributed tree species from European temperate regions. Although none of the tracers entered the leaf through the stomatal pores, small amounts of silver precipitation were observed in some epidermal cells, indicating moderate cuticular uptake. Trichomes, however, were shown to absorb and redistribute considerable amounts of ionic and fluorescent tracers. Moreover, microcomputed tomography indicated that 72% of empty trichomes refilled during leaf surface wetting and microscopic investigations revealed that trichomes do not have a cuticle but are covered with a pectin-rich cell wall layer. Taken together, our findings demonstrate that foliar trichomes, which exhibit strong hygroscopic properties as a result of their structural and chemical design, constitute a major FWU pathway in beech.

Extreme summer heat and drought lead to early fruit abortion in European beech.

Years with high fruit production, known as mast years, are the usual reproduction strategy of European beech. Harsh weather conditions such as frost during flowering can lead to pollination failure in spring. It has been assumed that mast is controlled by flowering, and that after successful pollination, high amounts of fruits and seeds would be produced. However, the extremely hot and dry European summer of 2018 showed that despite successful pollination, beechnuts did not develop or were only abundant in a few forest stands. An in-depth analysis of three forest sites of European beech from the Swiss Long-Term Forest Ecosystem Research Programme over the last 15-19 years revealed for the first time that extreme summer heat and drought can act as an "environmental veto", leading to early fruit abortion. Within the forest stands in years with fruit abortion, summer mean temperatures were 1.5 °C higher and precipitation sums were 45% lower than the long-term average. Extreme summer heat and drought, together with frost during flowering, are therefore disrupting events of the assumed biennial fruiting cycle in European beech.

Identification of a cyanidin-3-O-ß-galactoside in gall tissue induced by midges on Japanese beech (Fagus crenata Blume).

Insect gall structures have many characteristic forms and colors, which are distinguishable from host plants. In this study, we identified an anthocyanin from red color insect galls and revealed that the anthocyanin biosynthesis of plants was induced by the gall extracts. The galling insects presumably regulate the anthocyanin biosynthesis of host plants to protect their larvae from environmental stresses.

Regulation of thiol metabolism as a factor that influences the development and storage capacity of beech seeds.

Seeds are the basis of propagation for the common beech (Fagus sylvatica L.), but the seed set of the beech is unsteady, with 5-10 years between abundant crops. Beech seeds are very difficult to store and lose their viability quickly even in optimum storage conditions. To date, it has not been possible to determine factors indicative of the aging process and the loss of viability of beech seeds during storage. To address this important economic challenge and interesting scientific problem, we analyzed the adjustment of the redox state during the development and storage of seeds. Many metabolic processes are based on reduction and oxidation reactions. Thiol proteins control and react to the redox state in the cells. The level of thiol proteins increased during seed maturation and decreased during storage. Gel-based redox proteomics identified 17 proteins in beech seeds during development. The proteins could be assigned to processes like metabolism and antioxidant functions. During storage, the number of proteins decreased to only six, i.e., oxidoreductases, peptidases, hydrolases and isomerases. The occurrence of peroxiredoxins (PRX) as thiol peroxidases and redox regulators indicates an important role of cytosolic 1CysPRX and PRXIIC, mitochondrial PRXIIF, and plastidic PRXIIE, 2CysPRX, and PRXQ in beech seeds during development and storage. Particularly, 2CysPRX was present in beech seeds during development and storage and may perform an important function in regulation of the redox state during both seed development and storage. The role of thiol proteins in the regulation of the redox state during the development and storage of beech seeds is discussed.

Differences in carbon isotope leaf-to-phloem fractionation and mixing patterns along a vertical gradient in mature European beech and Douglas fir.

While photosynthetic isotope discrimination is well understood, the postphotosynthetic and transport-related fractionation mechanisms that influence phloem and subsequently tree ring δ13 C are less investigated and may vary among species. We studied the seasonal and diel courses of leaf-to-phloem δ13 C differences of water-soluble organic matter (WSOM) in vertical crown gradients and followed the assimilate transport via the branches to the trunk phloem at breast height in European beech (Fagus sylvatica) and Douglas fir (Pseudotsuga menziesii). δ13 C of individual sugars and cyclitols from a subsample was determined by compound-specific isotope analysis. In beech, leaf-to-phloem δ13 C differences in WSOM increased with height and were partly caused by biochemical isotope fractionation between leaf compounds. 13 C-Enrichment of phloem sugars relative to leaf sucrose implies an additional isotope fractionation mechanism related to leaf assimilate export. In Douglas fir, leaf-to-phloem δ13 C differences were much smaller and isotopically invariant pinitol strongly influenced leaf and phloem WSOM. Trunk phloem WSOM at breast height reflected canopy-integrated δ13 C in beech but not in Douglas fir. Our results demonstrate that leaf-to-phloem isotope fractionation and δ13 C mixing patterns along vertical gradients can differ between tree species. These effects have to be considered for functional interpretations of trunk phloem and tree ring δ13 C.

The impact of prolonged drought on phloem anatomy and phloem transport in young beech trees.

Phloem failure has recently been recognized as one of the mechanisms causing tree mortality under drought, though direct evidence is still lacking. We combined 13C pulse-labelling of 8-year-old beech trees (Fagus sylvatica L.) growing outdoors in a nursery with an anatomical study of the phloem tissue in their stems to examine how drought alters carbon transport and phloem transport capacity. For the six trees under drought, predawn leaf water potential ranged from -0.7 to -2.4 MPa, compared with an average of -0.2 MPa in five control trees with no water stress. We also observed a longer residence time of excess 13C in the foliage and the phloem sap in trees under drought compared with controls. Compared with controls, excess 13C in trunk respiration peaked later in trees under moderate drought conditions and showed no decline even after 4 days under more severe drought conditions. We estimated higher phloem sap viscosity in trees under drought. We also observed much smaller sieve-tube radii in all drought-stressed trees, which led to lower sieve-tube conductivity and lower phloem conductance in the tree stem. We concluded that prolonged drought affected phloem transport capacity through a change in anatomy and that the slowdown of phloem transport under drought likely resulted from a reduced driving force due to lower hydrostatic pressure between the source and sink organs.

Ultraviolet radiation modulates C:N stoichiometry and biomass allocation in Fagus sylvatica saplings cultivated under elevated CO2 concentration.

Under the conditions of ongoing climate change, terrestrial ecosystems will be simultaneously exposed to a permanent rise in atmospheric CO2 concentration and increasing variability of such environmental factors as temperature, precipitation, and UV radiation. This will result in numerous interactions. The interactive effects caused by exposure to such multiple environmental factors are not yet well understood. We tested the hypotheses that enhanced UV radiation reduces the stimulatory effect of elevated CO2 concentration on plant biomass production and that it alters biomass allocation in broadleaved European beech (Fagus sylvatica L.) saplings. Our results after 2 years of exposure confirmed interactive effects of CO2 concentration and UV radiation on biomass production, and particularly on biomass allocation to roots and aboveground biomass. The strongest stimulatory effect of elevated CO2 on aboveground biomass and roots was found under ambient UV radiation, while both low and high UV doses reduced this stimulation. Nitrogen content in the roots and the distribution of nitrogen among leaves and roots were also significantly affected by interaction of CO2 concentration and UV radiation. The observed changes in leaf and root C:N stoichiometry were associated with altered morphological traits, and particularly with a change in the proportion of fine roots. As the biomass allocation and especially the proportion of fine roots can play an important role in effective water and nutrient use and acclimation to future climates, it is essential to obtain a deeper understanding of the links between C:N stoichiometry and biomass accumulation.

Repeated summer drought delays sugar export from the leaf and impairs phloem transport in mature beech.

Phloem sustains maintenance and growth processes through transport of sugars from source to sink organs. Under low water availability, tree functioning is impaired, i.e., growth/photosynthesis decline and phloem transport may be hindered. In a 3-year throughfall exclusion (TE) experiment on mature European beech (Fagus sylvatica L.) we conducted 13CO2 branch labeling to investigate translocation of recently fixed photoassimilates under experimental drought over 2 years (2015 and 2016). We hypothesized (H1) that mean residence time of photoassimilates in leaves (MRT) increases, whereas (H2) phloem transport velocity (Vphloem) decreases under drought. Transport of carbohydrates in the phloem was assessed via δ13C of CO2 efflux measured at two branch positions following 13CO2 labeling. Pre-dawn water potential (ΨPD) and time-integrated soil water deficit (iSWD) were used to quantify drought stress. The MRT increased by 46% from 32.1 ± 5.4 h in control (CO) to 46.9 ± 12.3 h in TE trees, supporting H1, and positively correlated (P < 0.001) with iSWD. Confirming H2, Vphloem in 2016 decreased by 47% from 20.7 ± 5.8 cm h-1 in CO to 11.0 ± 2.9 cm h-1 in TE trees and positively correlated with ΨPD (P = 0.001). We suggest that the positive correlation between MRT and iSWD is a result of the accumulation of osmolytes maintaining cell turgor in the leaves under longer drought periods. Furthermore, we propose that the positive correlation between Vphloem and ΨPD is due to a lower water uptake of phloem conduits from surrounding tissues under increasing drought leading to a higher phloem sap viscosity and lower Vphloem. The two mechanisms increasing MRT and reducing Vphloem respond differently to low water availability and impair trees' carbon translocation under drought.