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Tree rings have been reliably used as an environmental proxy over the past decades for environmental reconstructions, simulations and forecasting. In our study, we investigated whether tree-ring chronologies are impacted by pollution. We chose sites in the Krušné hory and the Krkonoše Mountains in the Czech Republic which have a known history of pollution. We sampled Norway spruce (Picea abies [L.] Karst) in both ranges and compared their chronologies. We found no significant difference in the overall radial growth in the chronologies from both regions. However, we observed an increased heterogeneity in the growth of trees from the 1970s till the 1990s. Coherently, a severe reduction in tree growth from the late 1970s and a recovery towards the early 1990s was evident. We collected and analysed soil samples for pH and exchangeable element concentrations. All seven sampling sites' soils were strongly acidic (pH CaCl2  = 3.3 ± 0.4). The average soil base saturation at Krušné hory was higher than at Krkonoše (39% versus 12%), likely due to more intensive liming. Further, we compared these chronologies to other sites in Europe. Analysing 89 sites, we found that most (9 out of 14) of the sites with significantly reduced radial tree growth were located within the former 'Black Triangle', an area which was subjected to heavy industrialisation and pollution from the 1960s to the 1990s. Atmospheric sulphur deposition was found to negatively affect radial tree-growth, while limited quantities of oxidised nitrogen appeared to have a positive effect. Our results are consistent with previous research, indicating that atmospheric SO 2 pollution and subsequent acid fog and rime have led to a reduction in annual radial tree growth across the Black Triangle., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Introduction Acetylsalicylic acid (ASA) has commonly been touted for its potential to extend the aesthetic lifespan (EL) of Christmas trees when added to the water in the tree stand. This study examined the efficacy of ASA in prolonging the aesthetic longevity of spruce branches, in comparison to placebo and sildenafil. Intervention We conducted a triple-blinded, randomised clinical trial, wherein 60 spruce branches were allocated to one of three treatment arms in a 1:1:1 ratio. The primary intervention was ASA, compared against both placebo and sildenafil treatments. The study's primary endpoint was the EL of the spruce branches. Results All participating branches completed the study. No statistically significant differences were observed in the survival times across the three groups: ASA 17 days (standard deviation (SD): 6), placebo 20 days (SD: 8), and sildenafil 21 days (SD: 7); p = 0.30. Both the log-rank test and adjusted Cox proportional-hazards analyses failed to show any significant variations in aesthetic survival time among the treatment arms (p > 0.05). Conclusion Given our findings, there is no empirical support for the widely held recommendation of adding ASA to the water at the base of a Christmas tree to extend its aesthetic lifespan. Funding none. Trial registration none., (Published under Open Access CC-BY-NC-BD 4.0. https://creativecommons.org/licenses/by-nc-nd/4.0/.)

In response to various stimuli, plants acquire resistance against pests and/or pathogens. Such acquired or induced resistance allows plants to rapidly adapt to their environment. Spraying the bark of mature Norway spruce (Picea abies) trees with the phytohormone methyl jasmonate (MeJA) enhances resistance to tree-killing bark beetles and their associated phytopathogenic fungi. Analysis of spruce chemical defenses and beetle colonization success suggests that MeJA treatment both directly induces immune responses and primes inducible defenses for a faster and stronger response to subsequent beetle attack. We used metabolite and transcriptome profiling to explore the mechanisms underlying MeJA-induced resistance in Norway spruce. We demonstrated that MeJA treatment caused substantial changes in the bark transcriptional response to a triggering stress (mechanical wounding). Profiling of mRNA expression showed a suite of spruce inducible defenses are primed following MeJA treatment. Although monoterpenes and diterpene resin acids increased more rapidly after wounding in MeJA-treated than control bark, expression of their biosynthesis genes did not. We suggest that priming of inducible defenses is part of a complex mixture of defense responses that underpins the increased resistance against bark beetle colonization observed in Norway spruce. This study provides the most detailed insights yet into the mechanisms underlying induced resistance in a long-lived gymnosperm., (© 2020 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Little is known about how forests adjust their gas-exchange mode while atmospheric CO 2 rises globally and air quality changes regionally. The present study aims at addressing this research gap for boreal spruce trees growing in three different regions of Canada, submitted to distinct levels of atmospheric emissions, by examining the amount of carbon gained per unit of water lost in trees, i.e., the intrinsic water use efficiency (iWUE). Under pristine air quality conditions, middle-to long-term trends passed from no-reaction mode to passive strategies due to atmospheric CO 2 , and short-term iWUE variations mostly ensue from year-to-year climatic conditions. In contrast, in trees exposed to pollutants from a copper smelter and an oil-sands mining region, air quality deterioration generated swift, long-term iWUE rises immediately at the onset of operations. In this case, the very active foliar strategy sharply reduced the intra-foliar CO 2 (Ci) pressure. Statistical modeling allowed identifying emissions as the main trigger for the iWUE swift shifts; subsequent combined effects of emissions and rising CO 2 led to passive foliar modes in the recent decades, and short-term variations due to climatic conditions appeared all along the series. Overall, boreal trees under different regional conditions modified their foliar strategies mostly without changing their stem growth. These findings underline the potential of acidifying emissions for prompting major iWUE increases due to lowering the stomatal apertures in leaves, and the combined influence of rising CO 2 in modulating other foliar responses. A fallout of this research is that degrading air quality may create true divergences in the relationship between tree-ring isotopes and climatic conditions, an impact to consider prior to using isotopic series for paleo-climatic modeling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Natural Resources Canada. Published by Elsevier Ltd.. All rights reserved.)

The effects of auxins 2,4-D (2,4-dichlorophenoxyacetic acid), NAA (1-naphthaleneacetic acid) or picloram (4-amino-3,5,6-trichloropicolinic acid; 9 µM) and cytokinin BA (benzyloadenine; 4.5 µM) applied in the early stages of somatic embryogenesis (SE) on specific stages of SE in Picea abies and P. omorika were investigated. The highest SE initiation frequency was obtained after 2,4-D application in P. omorika (22.00%) and picloram application in P. abies (10.48%). NAA treatment significantly promoted embryogenic tissue (ET) proliferation in P . abies , while 2,4-D treatment reduced it. This reduction was related to the oxidative stress level, which was lower with the presence of NAA in the proliferation medium and higher with the presence of 2,4-D. The reduced oxidative stress level after NAA treatment suggests that hydrogen peroxide (H 2 O 2 ) acts as a signalling molecule and promotes ET proliferation. NAA and picloram in the proliferation medium decreased the further production and maturation of P. omorika somatic embryos compared with that under 2,4-D. The quality of the germinated P. abies embryos and their development into plantlets depended on the auxin type and were the highest in NAA-originated embryos. These results show that different auxin types can generate different physiological responses in plant materials during SE in both spruce species., Competing Interests: The authors declare no conflict of interest.

Community-scale impacts of glyphosate-based herbicides on wetland plant communities and the magnitude of those impacts that should be considered biologically relevant are poorly understood. We contrast three different thresholds for setting biologically meaningful critical effect sizes for complex ANOVA study designs. We use each of the of the critical effect sizes to determine optimal α levels for assessment of how different concentrations of glyphosate-based herbicides affect wetland plant communities over two years of herbicide application (alone and in combination with agricultural fertilizers) and two subsequent years without herbicide (or fertilizer) application. The application of glyphosate-based herbicides was found to result in a decrease in macrophyte species richness, an increase in macrophyte species evenness, a decrease in macrophyte cover and a reduction in community similarity. There was little evidence that nutrient additions directly or indirectly affected plant community endpoints. The glyphosate effects were evident in the first year of herbicide application in 2009, and became more pronounced in the second year of herbicide application in 2010. However, when herbicides were not applied in 2011, recovery was observed in most endpoints, with the exception being species evenness, for which partial recovery was not observed until 2012. Optimal α levels differed among the three critical effect sizes for each ANOVA term and endpoint combination, however regardless of differences in α levels, conclusions were generally consistent across all critical effect sizes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Although plants are often exposed to atmospheric nanoparticles (NPs), the mechanism of NP deposition and their effects on physiology and metabolism, and particularly in combination with other stressors, are not yet understood. Exploring interactions between stressors is particularly important for understanding plant responses in urban environments where elevated temperatures can be associated with air pollution. Accordingly, 3-year-old spruce seedlings were exposed for 2 weeks to aerial cadmium oxide (CdO) NPs of environmentally relevant size (8-62 nm) and concentration (2 × 10 5  cm -3 ). While half the seedlings were initially acclimated to high temperature (35 °C) and vapour pressure deficit (VPD; 2.81 kPa), the second half of the plants were left under non-stressed conditions (20 °C, 0.58 kPa). Atomic absorption spectrometry was used to determine Cd content in needles, while gas and liquid chromatography was used to determine changes in primary and secondary metabolites. Photosynthesis-related processes were explored with gas-exchange and chlorophyll fluorescence systems. Our work supports the hypothesis that atmospheric CdO NPs penetrate into leaves but high temperature and VPD reduce such penetration due to stomatal closure. The hypothesis that atmospheric CdO NPs influences physiological and metabolic processes in plants was also confirmed. This impact strengthens with increasing time of exposure. Finally, we found evidence that plants acclimated to stress conditions have different sensitivity to CdO NPs compared to plants not so acclimated. These findings have important consequences for understanding impacts of global warming on plants and indicates that although the effects of elevated temperatures can be deleterious, this may limit other forms of plant stress associated with air pollution., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Abscisic acid (ABA) is one of the main participants in the regulation of plant responses to water deficiency. Knowledge of the ABA signal transduction pathways in gymnosperms is rather limited, especially in comparison with those in angiosperms. Seedlings of Scots pine and Norway spruce are known for their contrasting behaviour strategies under water deficit. To characterize the possible role of ABA in these differences, ABA dynamics were investigated under conditions of water deficit in seedlings of these two species. The content of ABA and its catabolites was followed in the roots and needles of seedlings of Pinus sylvestris and Picea abies under conditions of polyethylene glycol (PEG)-induced water deficiency (-0.15 and -0.5 MPa) for 10 days. The expression of the main genes for ABA-biosynthetic enzymes was also analysed. ABA showed more pronounced stress-dependent dynamics in pine roots than in spruce roots, whereas in needles, the response was greater for spruce than pine. The ABA increase during drought was mainly due to de novo synthesis and the shift in the balance between ABA synthesis and catabolism towards synthesis. The ABA-glucosyl ester did not serve as a reserve for the release of free ABA under water deficiency. The expression levels of the main ABA biosynthetic genes showed a weak or no correlation with changes in ABA content under water stress, i.e., the ABA content in the seedlings of both species was not directly linked to the transcript levels of the main ABA biosynthetic genes. Less-pronounced stress-induced changes in ABA in pine needles than in spruce needles may be related to pine seedlings having a less conservative strategy of growth and maintenance of water balance under water deficit., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Somatic embryogenesis is an important biotechnological technique which can be used in studies associated with environmental stress. Four embryogenic cell lines of Norway spruce were grown on media enriched with copper and arsenic in concentration ranges 50-500 μM and 10-50 μM, respectively. The effects were observed during subsequent stages of somatic embryogenesis, the characteristics evaluated being proliferation potential, average number of somatic embryos obtained per g/fresh weight, morphology of developed somatic embryos, metal uptake, and microanalysis of macro- and micronutrients uptake. Copper and arsenic at higher concentrations significantly reduced the growth of early somatic embryos. In almost all treatments, the cell line V-1-3 showed the best performance compared with the other lines tested. Environmental scanning electron microscopy was used to visualize and identify morphological abnormalities in the development of somatic embryos. Abnormalities observed were classified into several categories: meristemless somatic embryos, somatic embryos with disrupted meristem, reduced number of cotyledons, single cotyledon and fused cotyledons. With the application of a low temperature method for the environmental scanning electron microscope, samples were stabilized and whole meristems could be investigated in their native state. As far as we are aware, this is the first report of the effect of copper and arsenic during the process of somatic embryogenesis and the first to evaluate the content of macro and micronutrients uptake in Norway spruce., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In the 45 years after legislation of the Clean Air Act, there has been tremendous progress in reducing acidic air pollutants in the eastern United States, yet limited evidence exists that cleaner air has improved forest health. Here, we investigate the influence of recent environmental changes on the growth and physiology of red spruce (Picea rubens Sarg.) trees, a key indicator species of forest health, spanning three locations along a 100 km transect in the Central Appalachian Mountains. We incorporated a multiproxy approach using 75-year tree ring chronologies of basal tree growth, carbon isotope discrimination (∆ 13 C, a proxy for leaf gas exchange), and δ 15 N (a proxy for ecosystem N status) to examine tree and ecosystem level responses to environmental change. Results reveal the two most important factors driving increased tree growth since ca. 1989 are reductions in acidic sulfur pollution and increases in atmospheric CO 2 , while reductions in pollutant emissions of NO x and warmer springs played smaller, but significant roles. Tree ring ∆ 13 C signatures increased significantly since 1989, concurrently with significant declines in tree ring δ 15 N signatures. These isotope chronologies provide strong evidence that simultaneous changes in C and N cycling, including greater photosynthesis and stomatal conductance of trees and increases in ecosystem N retention, were related to recent increases in red spruce tree growth and are consequential to ecosystem recovery from acidic pollution. Intrinsic water use efficiency (iWUE) of the red spruce trees increased by ~51% across the 75-year chronology, and was driven by changes in atmospheric CO 2 and acid pollution, but iWUE was not linked to recent increases in tree growth. This study documents the complex environmental interactions that have contributed to the recovery of red spruce forest ecosystems from pervasive acidic air pollution beginning in 1989, about 15 years after acidic pollutants started to decline in the United States., (© 2018 The Authors. Global Change Biology Published by John Wiley & Sons Ltd.)

High latitude forests will experience large changes in temperature and CO 2 concentrations this century. We evaluated the effects of future climate conditions on 2 dominant boreal tree species, Pinus sylvestris L. and Picea abies (L.) H. Karst, exposing seedlings to 3 seasons of ambient (430 ppm) or elevated CO 2 (750 ppm) and ambient temperatures, a + 4 °C warming or a + 8 °C warming. Pinus sylvestris responded positively to warming: seedlings developed a larger canopy, maintained high net CO 2 assimilation rates (A net ), and acclimated dark respiration (R dark ). In contrast, carbon fluxes in Picea abies were negatively impacted by warming: maximum rates of A net decreased, electron transport was redirected to alternative electron acceptors, and thermal acclimation of R dark was weak. Elevated CO 2 tended to exacerbate these effects in warm-grown Picea abies, and by the end of the experiment Picea abies from the +8 °C, high CO 2 treatment produced fewer buds than they had 3 years earlier. Treatments had little effect on leaf and wood anatomy. Our results highlight that species within the same plant functional type may show opposite responses to warming and imply that Picea abies may be particularly vulnerable to warming due to low plasticity in photosynthetic and respiratory metabolism., (© 2018 John Wiley & Sons Ltd.)

With the constant accumulation of polycyclic aromatic hydrocarbons (PAHs) in soil and increasing temperature and CO 2 levels, plants will inevitably be exposed to combined stress. Studies on the effects of such combined stresses are needed to develop mitigation and adaptation measures. Here, we investigated the effects of soil pyrene contamination (50 mg kg -1 ) on growth and phenolics of 1-year-old Norway spruce seedlings from five different origins in Finland at elevated temperature (+ 2 °C) and CO 2 (+ 360 ppm). Pyrene significantly decreased spruce height growth (0-48%), needle biomass (0-44%), stem biomass (0-43%), and total phenolic concentrations in needles (2-13%) and stems (1-19%) compared to control plants. Elevated temperature alone did not affect growth but led to lower concentrations of total phenolics in needles (5-29%) and stems (5-18%) in both soil treatments. By contrast, elevated CO 2 led to higher needle biomass (0-39%) in pyrene-spiked soils and higher concentrations of stem phenolics (0-18%) in pyrene-spiked and control soils compared to ambient treatments. The decrease in height growth and phenolic concentrations caused by pyrene was greater at elevated temperature, while elevated CO 2 only marginally modified the response. Seedlings from different origins showed different responses to the combined environmental stressors. The changes in growth and in the quantity and quality of phenolics in this study suggest that future climate changes will aggravate the negative influence of soil pyrene pollution on northern conifer forest ecosystems.

We investigated fungal growth and community composition in buried meshbags, amended with apatite, biotite or hornblende, in Norway spruce (Picea abies) forests of varying nutrient status. Norway spruce needles and soil collected from forests overlying serpentinite had low levels of potassium and phosphorus, those from granite had low levels of magnesium, whereas those from amphibolite had comparably high levels of these nutrients. We assayed the fungal colonization of meshbags by measuring ergosterol content and fungal community with 454 sequencing of the internal transcribed spacer region. In addition, we measured fine root density. Fungal biomass was increased by apatite amendment across all plots and particularly on the K- and P-deficient serpentinite plots, whereas hornblende and biotite had no effect on fungal biomass on any plots. Fungal community (total fungal and ectomycorrhizal) composition was affected strongly by sampling location and soil depth, whereas mineral amendments had no effect on community composition. Fine root biomass was significantly correlated with fungal biomass. Ectomycorrhizal communities may respond to increased host-tree phosphorus demand by increased colonization of phosphorus-containing minerals, but this does not appear to translate to a shift in ectomycorrhizal community composition. This growth response to nutrient demand does not appear to exist for potassium or magnesium limitation., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

A dynamic coupled biogeochemical-ecological model was used to simulate the effects of nitrogen deposition and climate change on plant communities at three forest sites in France. The three sites had different forest covers (sessile oak, Norway spruce and silver fir), three nitrogen loads ranging from relatively low to high, different climatic regions and different soil types. Both the availability of vegetation time series and the environmental niches of the understory species allowed to evaluate the model for predicting the composition of the three plant communities. The calibration of the environmental niches was successful, with a model performance consistently reasonably high throughout the three sites. The model simulations of two climatic and two deposition scenarios showed that climate change may entirely compromise the eventual recovery from eutrophication of the simulated plant communities in response to the reductions in nitrogen deposition. The interplay between climate and deposition was strongly governed by site characteristics and histories in the long term, while forest management remained the main driver of change in the short term., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Two unrelated, geographically distinct, highly embryogenic lines of Norway spruce (Picea abies (L.) Karst.) were analysed to identify metabolic traits characteristic for lines with good yields of high-quality embryos. The results were compared with corresponding characteristics of a poorly productive line (low embryo yield, scarce high-quality embryos). The following carbohydrate profiles and spectra during maturation, desiccation and germination were identified as promising characteristics for line evaluation: a gradual decrease in total soluble carbohydrates with an increasing sucrose : hexose ratio during maturation; accumulation of raffinose family oligosaccharides resulting from desiccation and their rapid degradation at the start of germination; and a decrease in sucrose, increase in hexoses and the appearance of pinitol with proceeding germination. We propose that any deviation from this profile in an embryonic line is a symptom of inferior somatic embryo development. We further propose that a fatty acid spectrum dominated by linoleic acid (18 : 2) was a common feature of healthy spruce somatic embryos, although it was quite different from zygotic embryos mainly containing oleic acid (18 : 1). The responses of the lines to osmotic stress were evaluated based on comparison of control (without osmoticum) and polyethylene glycol (PEG)-exposed (PEG 4000) variants. Although genetically distinct, both highly embryogenic lines responded in a very similar manner, with the only difference being sensitivity to high concentrations of PEG. At an optimum PEG concentration (3.75 and 5%), which was line specific, negative effects of PEG on embryo germination were compensated for by a higher maturation efficiency so that the application of PEG at an appropriate concentration improved the yield of healthy germinants per gram of initial embryonal mass and accelerated the process. Polyethylene glycol application, however, resulted in no improvement of the poorly productive line., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Histone deacetylase (HDAC) is a crucial component in the regulation of gene expression in various cellular processes in animal and plant cells. HDAC has been reported to play a role in embryogenesis. However, the effect of HDAC on androgamete development remains unclear, especially in gymnosperms. In this study, we used the HDAC inhibitors trichostatin A (TSA) and sodium butyrate (NaB) to examine the role of HDAC in Picea wilsonii pollen germination and pollen tube elongation. Measurements of the tip-focused Ca2+ gradient revealed that TSA and NaB influenced this gradient. Immunofluorescence showed that actin filaments were disrupted into disorganized fragments. As a result, the vesicle trafficking was disturbed, as determined by FM4-64 labeling. Moreover, the distribution of pectins and callose in cell walls was significantly altered in response to TSA and NaB. Our results suggest that HDAC affects pollen germination and polarized pollen tube growth in Picea wilsonii by affecting the intracellular Ca2+ concentration gradient, actin organization patterns, vesicle trafficking, as well as the deposition and configuration of cell wall components.

Background: Somatic embryogenesis in conifer species has great potential for the forestry industry. Hence, a number of methods have been developed for their efficient and rapid propagation through somatic embryogenesis. Although information is available regarding the previous process-mediated generation of embryogenic cells to form somatic embryos, there is a dearth of information in the literature on the detailed structure of these clusters., Methodology/principal Findings: The main aim of this study was to provide a more detailed structure of the embryogenic tissue clusters obtained through the in vitro propagation of the Norway spruce (Picea abies (L.) Karst.). We primarily focused on the growth of early somatic embryos (ESEs). The data on ESE growth suggested that there may be clear distinctions between their inner and outer regions. Therefore, we selected ESEs collected on the 56th day after sub-cultivation to dissect the homogeneity of the ESE clusters. Two colourimetric assays (acetocarmine and fluorescein diacetate/propidium iodide staining) and one metabolic assay based on the use of 2,3,5-triphenyltetrazolium chloride uncovered large differences in the metabolic activity inside the cluster. Next, we performed nuclear magnetic resonance measurements. The ESE cluster seemed to be compactly aggregated during the first four weeks of cultivation; thereafter, the difference between the 1H nuclei concentration in the inner and outer clusters was more evident. There were clear differences in the visual appearance of embryos from the outer and inner regions. Finally, a cluster was divided into six parts (three each from the inner and the outer regions of the embryo) to determine their growth and viability. The innermost embryos (centripetally towards the cluster centre) could grow after sub-cultivation but exhibited the slowest rate and required the longest time to reach the common growth rate. To confirm our hypothesis on the organisation of the ESE cluster, we investigated the effect of cluster orientation on the cultivation medium and the influence of the change of the cluster's three-dimensional orientation on its development. Maintaining the same position when transferring ESEs into new cultivation medium seemed to be necessary because changes in the orientation significantly affected ESE growth., Conclusions and Significance: This work illustrated the possible inner organisation of ESEs. The outer layer of ESEs is formed by individual somatic embryos with high metabolic activity (and with high demands for nutrients, oxygen and water), while an embryonal group is directed outside of the ESE cluster. Somatic embryos with depressed metabolic activity were localised in the inner regions, where these embryonic tissues probably have a very important transport function.

Background and Aims: Plants growing under elevated atmospheric CO2 concentrations often have reduced stomatal conductance and subsequently increased leaf temperature. This study therefore tested the hypothesis that under long-term elevated CO2 the temperature optima of photosynthetic processes will shift towards higher temperatures and the thermostability of the photosynthetic apparatus will increase., Methods: The hypothesis was tested for saplings of broadleaved Fagus sylvatica and coniferous Picea abies exposed for 4-5 years to either ambient (AC; 385 µmol mol(-1)) or elevated (EC; 700 µmol mol(-1)) CO2 concentrations. Temperature response curves of photosynthetic processes were determined by gas-exchange and chlorophyll fluorescence techniques., Key Results: Initial assumptions of reduced light-saturated stomatal conductance and increased leaf temperatures for EC plants were confirmed. Temperature response curves revealed stimulation of light-saturated rates of CO2 assimilation (Amax) and a decline in photorespiration (RL) as a result of EC within a wide temperature range. However, these effects were negligible or reduced at low and high temperatures. Higher temperature optima (Topt) of Amax, Rubisco carboxylation rates (VCmax) and RL were found for EC saplings compared with AC saplings. However, the shifts in Topt of Amax were instantaneous, and disappeared when measured at identical CO2 concentrations. Higher values of Topt at elevated CO2 were attributed particularly to reduced photorespiration and prevailing limitation of photosynthesis by ribulose-1,5-bisphosphate (RuBP) regeneration. Temperature response curves of fluorescence parameters suggested a negligible effect of EC on enhancement of thermostability of photosystem II photochemistry., Conclusions: Elevated CO2 instantaneously increases temperature optima of Amax due to reduced photorespiration and limitation of photosynthesis by RuBP regeneration. However, this increase disappears when plants are exposed to identical CO2 concentrations. In addition, increased heat-stress tolerance of primary photochemistry in plants grown at elevated CO2 is unlikely. The hypothesis that long-term cultivation at elevated CO2 leads to acclimation of photosynthesis to higher temperatures is therefore rejected. Nevertheless, incorporating acclimation mechanisms into models simulating carbon flux between the atmosphere and vegetation is necessary., (© The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Embryogenic cultures of Picea balfouriana, which is an important commercial species for reforestation in Southern China, easily lose their embryogenic ability during long-term culture. Embryogenic tissue that proliferated at lower concentrations (3.6 μM and 2.5 μM) of 6-benzylaminopurine (6-BAP) were more productive, and generated 113 ± 6 and 89 ± 3 mature embryos per 100 mg embryogenic tissue, respectively. A metabolomic approach was used to study the changes in metabolites linked to embryogenic competence related to three different 6-BAP concentrations (2.5 μM, 3.6 μM, and 5 μM). A total of 309 compounds were obtained, among which 123 metabolites mapped to Kyoto Encyclopedia of Genes and genomes (KEGG) pathways. The levels of 35 metabolites were significantly differentially regulated among the three 6-BAP treatments, and 32 metabolites differed between the 2.5 μM and 5 μM treatments. A total of 17 metabolites appeared only once among the three comparisons. The combination of a score plot and a loading plot showed that in the samples with higher embryogenic ability (3.6 μM and 2.5 μM), up-regulated metabolites were mostly amino acids and down-regulated metabolites were mostly primary carbohydrates (especially sugars). These results suggested that 6-BAP may influence embryogenic competence by nitrogen metabolism, which could cause an increase in amino acid levels and higher amounts of aspartate, isoleucine, and leucine in tissues with higher embryogenic ability. Furthermore, we speculated that 6-BAP may affect the amount of tryptophan in tissues, which would change the indole-3-acetic acid levels and influence the embryogenic ability.

The cytokinin 6-benzylaminopurine (6-BAP) influences the embryogenic capacity of the tissues of Picea balfouriana during long subculture (after 3 months). Tissues that proliferate in 3.6 and 5 µM 6-BAP exhibit the highest and lowest embryogenic capacity, respectively, generating 113 ± 6 and 23 ± 3 mature embryos per 100 mg of tissue. In this study, a comparative transcriptomic and proteomic approach was applied to characterize the genes and proteins that are differentially expressed among tissues under the influence of different levels of 6-BAP. A total of 51 375 unigenes and 2617 proteins were obtained after quality filtering. There were 2770 transcripts for proteins found among these unigenes. Gene ontology (GO) analysis of the differentially expressed unigenes and proteins showed that they were involved in cell and binding activity and were enriched in ribosome and glutathione metabolism pathways. Ribosomal proteins, glutathione S-transferase proteins, germin-like proteins and calmodulin-independent protein kinases were up-regulated in the embryogenic tissues with the highest embryogenic ability (treated with 3.6 µM 6-BAP), which was validated via quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and these proteins might serve as molecular markers of embryogenic ability. Data are available via Sequence Read Archive (SRA) and ProteomeXchange with identifier SRP042246 and PXD001022, respectively., (© 2014 Scandinavian Plant Physiology Society.)

The effect of long-term exposure of twice-ambient O(3) (2 × O(3)) on whole-tree nitrogen (N) uptake and partitioning of adult beech and spruce was studied in a mixed forest stand, SE-Germany. N uptake as (15)N tracer and N pools were calculated using N concentrations and biomass of tree compartments. Whole-tree N uptake tended to be lower under 2 × O(3) in both species compared to trees under ambient O(3) (1 × O(3)). Internal partitioning in beech showed significantly higher allocation of new N to roots, with mycorrhizal root tips and fine roots together receiving about 17% of new N (2 × O(3)) versus 7% (1 × O(3)). Conversely, in spruce, N allocation to roots was decreased under 2 × O(3). These contrasting effects on belowground N partitioning and pool sizes, being largely consistent with the pattern of N concentrations, suggest enhanced N demand and consumption of stored N with higher relevance for tree-internal N cycling in beech than in spruce., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Heavy metal pollution has became one of the realistic matters of globality. Previous reports indicated that heavy metals could significantly inhibit pollen germination and tube growth. In the present study, comparative studies on the effects of different heavy metals (As, Hg, Cd, Cr and Cu) on in-vitro picea wilsonii pollen germination and tube growth were carried out. Microscopic evaluation revealed that different heavy metals had various degree of toxicity on P. wilsonii pollen tube development. As showed the most toxic effects on pollen germination, which was followed by Hg and Cd, while Cr and Cu showed relatively lower toxicity. Besides, pollen tubes showed varying shapes in response to different heavy metal stress. Pollen tubes treated with Cd, Hg and As were usually characterized by irregularly increasing diameters and swelling tips with distinct cytoplasimic vacuolation. On the other hand, except for the slightly increased diameters, no obvious abnormal shape were observed in tubes treated with Cr or Cu. Lyso-Tracker Green staining indicated that only Cd-treated pollen tubes showed numerous vacuole-like acidic organelles, though cytoplasmic vacuolization were also observed in pollen tubes treated with Hg and A. In brief, our data indicated that different heavy metals have various effects on Picea wilsonii pollen germination and tube growth, and that in-vitro pollen culture might be used as a competent system for biomonitoring of air pollution.

Allocation of recent photoassimilates of juvenile beech and spruce in response to twice-ambient ozone (2 × O(3)) and plant competition (i.e. intra vs. inter-specific) was examined in a phytotron study. To this end, we employed continuous (13)CO(2)/(12)CO(2) labeling during late summer and pursued tracer kinetics in CO(2) released from stems. In beech, allocation of recent photoassimilates to stems was significantly lowered under 2 × O(3) and increased in spruce when grown in mixed culture. As total tree biomass was not yet affected by the treatments, C allocation reflected incipient tree responses providing the mechanistic basis for biomass partitioning as observed in longer experiments. Compartmental modeling characterized functional properties of substrate pools supplying respiratory C demand. Respiration of spruce appeared to be exclusively supplied by recent photoassimilates. In beech, older C, putatively located in stem parenchyma cells, was a major source of respiratory substrate, reflecting the fundamental anatomical disparity between angiosperm beech and gymnosperm spruce., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The estimate of growth losses by ozone exposure of forest trees is a significant part in current C sequestration calculations and will also be important in future modeling. It is therefore important to know if the relationship between ozone flux and growth reduction of young trees, used to derive a Critical Level for ozone, is also valid for mature trees. Epidemiological analysis of stem increment data from Fagus sylvatica L. and Picea abies Karst. observed in Swiss forest plots was used to test this hypothesis. The results confirm the validity of the flux-response relationship at least for beech and therefore enable estimating forest growth losses by ozone on a country-wide scale. For Switzerland, these estimates amount to 19.5% growth reduction for deciduous forests, 6.6% for coniferous forests and 11.0% for all forested areas based on annual ozone stomatal uptake during the time period 1991-2011., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In order to examine whether the leaves of the Colorado blue spruce (Picea pungens) are damaged or not by traffic pollution, the traits of the anatomy and physiology of its leaves are investigated by exposure to vehicle exhausts in a laboratory experiment lasting 30 days. The results show that both the anatomical structures and physiological traits of the leaves are significantly affected by vehicle exhausts. The anatomical structures, including epidermis, cuticle, palisade, and spongy parenchyma are modified when exposed to the high concentrations (≥ 0.4 mg/m(3)) of vehicle exhausts. However, physiological traits such as total chlorophyll content are not changed when exposed to different concentrations of vehicle exhaust. Unlike the total chlorophyll content, the electrical conductivities increased, whereas the POD activities decreased when presented in vehicle exhausts. The present study indicates that the Colorado blue spruce changes its anatomical structures and physiological traits to avoid possible damage by vehicle exhausts.

Sugars play an important role in various physiological processes during plant growth and development; however, the developmental roles and regulatory functions of hexoses other than glucose are still largely unclear. Recent studies suggest that blocked embryo development in Norway spruce (Picea abies (L.) Karst) is associated with accumulation of fructose. In the present study, the potential biochemical regulatory mechanism of glucose and fructose was studied during development of somatic embryos of Norway spruce from pro-embryogenic masses to mature embryos. The changes in protein fluorescence, a marker of the Maillard reaction, were monitored in two cell lines of Norway spruce that were grown on media containing sucrose (control), glucose or fructose. Manual time-lapse photography showed that growth of embryogenic cultures on medium containing sucrose was characterized by normal development of mature embryos whereas the embryogenic cultures that were grown on media containing glucose or fructose did not develop mature embryos. The biochemical analyses of embryogenic samples collected during embryo development showed that: (i) the content of glucose and fructose in the embryogenic cultures increased significantly during growth on each medium, respectively; (ii) the accumulation of Maillard products in the embryogenic cultures was highly correlated with the endogenous content of fructose but not glucose; and (iii) the embryogenic cultures grown on fructose displayed the highest protein carbonyl content and DNA damage whereas the highest content of glutathione was recorded in the embryogenic cultures that had grown on sucrose. Our data suggest that blocked development of embryos in the presence of fructose may be associated with the Maillard reaction., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The role of the actin cytoskeleton in somatic embryo development was investigated using latrunculin B and cytochalasin D. Brief treatments (1h) with either drug at the start of maturation fragmented the actin in suspensor cells and/or depolymerized actin filaments in meristematic cells. The drugs targeted different cells: latB primarily affected the suspensor cells, but cchD damaged both suspensor and meristematic cells. Lethal damage to the meristematic and suspensor cells was observed when the drugs were applied throughout the maturation period, although the severity of this effect depended on their concentrations. The drugs' effects on the yield of mature somatic embryos were investigated by applying them to embryo cultures throughout the maturation period or for one week at three different points in the maturation process: immediately prior to the start of maturation, during the first week of maturation, and during the fourth week of maturation. The strongest effects were observed when the drugs were applied at the start of maturation. Under these conditions, latB destroyed the suspensors, eliminating the underdeveloped embryos that depend on them. This accelerated the development of embryos that were capable of separating from the suspensors. Thus, while the total number of embryos at the end of the maturation period was lower than in untreated control cultures, the surviving mature embryos were of high quality. cchD treatment at the start of maturation strongly inhibited embryo development. Drug treatment at the end of the maturation period did not significantly affect embryo development: latB caused no change in the yield of somatic embryos, but cchD treatment increased the number of malformed embryos compared to untreated controls., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

As one of the most severe pollutants, cadmium has been reported to be harmful to plant cells, but the effects of cadmium on gymnosperm pollen germination and tube growth and the mechanism of this involvement are still unclear. Here, we report that cadmium not only strongly inhibited P. wilsonii pollen germination and tube growth, but also significantly altered tube morphology in a dose-dependent manner. Time-lapse images obtained with a laser scanning confocal microscope revealed that endocytosis was dramatically inhibited by cadmium stress. Further investigation with ER-Tracker dye indicated that cadmium stress reduced the number of the Golgi apparatus, and induced dilation of ER. Additionally, Lyso-Tracker staining showed that cadmium distinctly promoted the formation of acidic organelles in pollen tubes, likely derived from the dilated ER. Taken together, our studies indicated that P. wilsonii pollens were highly susceptible to cadmium stress, and that cadmium stress strongly inhibited pollen germination and tube growth by disrupting the endomembrane organelles, inhibiting endo/exocytosis, and forming acidic vacuoles, resulting in swollen tube tips and irregularly broadened tube diameters. These findings provide a new insight into the effects of cadmium toxicity on the tip growth of pollen tubes.

The effects of elevated ozone (1.4× ambient) and temperature (ambient +1.3 °C) alone and in combination were studied on the needle cell structure of soil-grown Norway spruce seedlings in the late growing season and winter. Temperature treatment continued over winter and lengthened the snow-free period. Elevated temperature caused microscopic changes related to photosynthesis (decreased chloroplast size and increased number), carbon storage (reduced starch and increased cytoplasmic lipids) and defence (decreased mitochondrial size and proportion per cytoplasm, increased peroxisomes and plastoglobuli, altered appearance of tannins). The results suggest increased oxidative stress by elevated temperature and altered allocation of limited carbon reserve to defence. The number of peroxisomes and plastoglobuli remained high in the outer cells of needles of ozone-exposed seedlings but decreased in the inner cells. This may indicate defence allocation to cells close to the stomata and surface, which are experiencing more oxidative stress. Ozone reduced winter hardiness based on seasonal changes in chloroplast shape and location in the cells. The effects of ozone became evident at the end of the growing season, indicating the effect of cumulative ozone dose or that the seedlings were vulnerable to ozone at the later phases of winter hardening. Elevated temperature increased cellular damage in early winter and visible damage in spring, and the damage was enhanced by ozone. In conclusion, the study suggests that modest air temperature elevation increases stress at the cell structural level in spruce seedlings and is enhanced by low ozone elevation. Future climatic conditions where snow cover is formed later or is lacking but temperatures are low can increase the risk of severe seedling damage, and current and future predicted ozone concentrations increase this risk.

A range of Organosolv pretreatments, using ethanol:water mixtures with dilute sulphuric acid, were applied to Sitka spruce sawdust with the aim of generating useful co-products as well as improving saccharification yield. The most efficient of the pretreatment conditions, resulting in subsequent saccharification yields of up to 86%, converted a large part of the hemicellulose sugars to their ethyl glycosides as identified by GC/MS. These conditions also reduced conversion of pentoses to furfural, the ethyl glycosides being more stable to dehydration than the parent pentoses. Through comparison with the behaviour of model compounds under the same reaction conditions it was shown that the anomeric composition of the products was consistent with a predominant transglycosylation reaction mechanism, rather than hydrolysis followed by glycosylation. The ethyl glycosides have potential as intermediates in the sustainable production of high-value chemicals., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

γ-Aminobutyric acid (GABA) is a four-carbon non-protein amino acid found in a wide range of organisms. Recently, GABA accumulation has been shown to play a role in the stress response and cell growth in angiosperms. However, the effect of GABA deficiency on pollen tube development remains unclear. Here, we demonstrated that specific concentrations of exogenous GABA stimulated pollen tube growth in Picea wilsonii, while an overdose suppressed pollen tube elongation. The germination percentage of pollen grains and morphological variations in pollen tubes responded in a dose-dependent manner to treatment with 3-mercaptopropionic acid (3-MP), a glutamate decarboxylase inhibitor, while the inhibitory effects could be recovered in calcium-containing medium supplemented with GABA. Using immunofluorescence labeling, we found that the actin cables were disorganized in 3-MP treated cells, followed by the transition of endo/exocytosis activating sites from the apex to the whole tube shank. In addition, variations in the deposition of cell wall components were detected upon labeling with JIM5, JIM7, and aniline blue. Our results demonstrated that calcium-dependent GABA signaling regulates pollen germination and polarized tube growth in P. wilsonii by affecting actin filament patterns, vesicle trafficking, and the configuration and distribution of cell wall components.

Alkaline oxidation pretreatment was developed for spruce, birch and sugar cane bagasse. The reaction was carried out in alkaline water solution under 10 bar oxygen pressure and at mild reaction temperature of 120-140°C. Most of the lignin was solubilised by the alkaline oxidation pretreatment and an easily hydrolysable carbohydrate fraction was obtained. After 72 h hydrolysis with a 10 FPU/g enzyme dosage, glucose yields of 80%, 91%, and 97%, for spruce, birch and bagasse, respectively, were achieved. The enzyme dosage could be decreased to 4 FPU/g without a major effect in terms of the hydrolysis performance. Compared to steam explosion alkaline oxidation was found to be significantly better in the conditions tested, especially for the pretreatment of spruce. In hydrolysis and fermentation at 12% d.m. consistency an ethanol yield of 80% could be obtained with both bagasse and spruce in 1-3 days., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Addition of sodium borohydride to a lignocellulose hydrolysate of Norway spruce affected the fermentability when cellulosic ethanol was produced using Saccharomyces cerevisiae. Treatment of the hydrolysate with borohydride improved the ethanol yield on consumed sugar from 0.09 to 0.31 g/g, the balanced ethanol yield from 0.02 to 0.30 g/g, and the ethanol productivity from 0.05 to 0.57 g/(L×h). Treatment of a sugarcane bagasse hydrolysate gave similar results, and the experiments indicate that sodium borohydride is suitable for chemical in situ detoxification. The model inhibitors coniferyl aldehyde, p-benzoquinone, 2,6-dimethoxybenzoquinone, and furfural were efficiently reduced by treatment with sodium borohydride, even under mild reaction conditions (20 °C and pH 6.0). While addition of sodium dithionite to pretreatment liquid from spruce improved enzymatic hydrolysis of cellulose, addition of sodium borohydride did not. This result indicates that the strong hydrophilicity resulting from sulfonation of inhibitors by dithionite treatment was particularly important for alleviating enzyme inhibition., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The saccharification of softwood using alkaline polyol pulping (AlkaPolP) and enzymatic hydrolysis was investigated. It will be demonstrated that the AlkaPolP process yields high quality pulps which can easily be hydrolyzed by cellulases. Temperature (180-230°C) and duration (15-60 min) of the alkaline glycerol pulping, expressed as pulping severity R0, were varied to find optimum reaction conditions. The obtained pulps were characterized regarding their residual lignin content, kappa number and crystallinity index. Thus, the dependencies of the conversion during enzymatic hydrolysis on severity, pulp composition and pulp characteristics could be observed. In further experiments it was investigated how the enzymatic hydrolysis is affected by pulp drying or by a reduction of enzyme loading. Up to 83% of the initial cellulose in wood and almost 97% of the cellulose in pulp were converted into glucose using cellulases from Trichoderma reesei and β-glucosidase from Aspergillus niger., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Wood samples (Picea jezoensis Carr.) were treated with solutions of aqueous NaOH (0-0.20 concentration fraction) and each treated samples evaluated by dynamic mechanical analyses (DMA). NaOH treatment was shown to affect the interactions between microfibrils and the surrounding matrix and, in particular, the dynamics of methylol groups in the microfibrils. The former is not dependent on the degree of crystallization but rather on the eluviation of the matrix. The latter depends on the degree of crystallization. Alkali treatment induces changes in the polymer domains as a result of matrix eluviation. This decreases the dynamics of methylol groups at NaOH concentrations less than 0.11. On the other hand, alkali treatment causes non-crystallization at concentrations greater than 0.11, which quantitatively increases the flexibility of methylol groups. Crystallinity decreased, and main-chain dynamics increased, following treatment with highly concentrated NaOH solutions. The dynamics of lignin also increased due to weakened interactions with microfibrils due to non-crystallization., (Copyright © 2012 Elsevier B.V. All rights reserved.)

According to well-known biochemical and biophysical mechanisms, the stimulation of C(3) photosynthesis by elevated atmospheric CO(2) concentration ([CO(2)]) is strongly modified by changes in temperature and radiation. In order to investigate whether a static parameterization of the commonly used Farquhar et al. model of photosynthesis (i.e., without CO(2)-induced seasonal or thermal acclimation of photosynthetic capacity) can accurately predict these interactions in mature boreal Norway spruce (Picea abies (L.) Karst.) during the frost-free part of the growing season, shoot gas exchange was continuously measured on trees during their second/third year of exposure to ambient or doubled [CO(2)] inside whole-tree chambers. The relative CO(2)-induced enhancement of net photosynthesis (A(n)) at a given temperature remained stable over the study period, but increased strongly with temperature and radiation, in agreement with predictions by the model. Light-saturated A(n) (+67% at 20 °C), dark respiration (+36%) and intercellular to ambient [CO(2)] ratio (c(i)/c(a); +27%) were significantly increased by CO(2) treatment. Stomatal conductance (g(s)) was not significantly affected. Our results demonstrate that the Farquhar et al. model of photosynthesis has the capability to predict interactions between [CO(2)] and seasonal weather variability on A(n) in Norway spruce during the non-frost growing season without accounting for CO(2)-induced seasonal and/or thermal photosynthetic acclimation. However, stomatal model assumptions of reduced g(s) and constant c(i)/c(a) under rising atmospheric [CO(2)] did not hold.

Ectomycorrhizal (ECM) fungi are efficient at taking up phosphorus (P) from mineral sources, such as apatite, which are not easily available to the host trees. Since ECM fungal species differ in P uptake rates, it can be expected that the composition of the ECM fungal community will change upon exposure to apatite, provided that the P transfer is rewarded by more carbon being transferred to the fungal symbiont. Control and apatite-amended mesh bags were buried in pairs in the humus layer of a P-poor Norway spruce forest. The ECM fungal community that colonized these bags was analyzed by DNA extraction, PCR amplification of the internal transcribed spacer (ITS) region, cloning, and random sequencing. Fungal biomass was estimated by ergosterol analysis. No change in the ECM fungal community structure was seen after 5 years of apatite exposure, although the fungal biomass increased threefold upon apatite amendment. Our results indicate that host trees enhance carbon allocation to ECM fungi colonizing P sources in P-poor forests but the lack of change in the composition of the ECM fungal community suggests that P transfer rates were similar among the species. Alternatively, higher P transfer among certain species was not rewarded with higher carbon transfer from the host.

Background: Plant defensins represent a major innate immune protein superfamily that displays strong inhibitory effects on filamentous fungi. The total number of plant defensins in a conifer species is unknown since there are no sequenced conifer genomes published, however the genomes of several angiosperm species provide an insight on the diversity of plant defensins. Here we report the identification of five new defensin-encoding genes from the Picea glauca genome and the characterization of two of their gene products, named PgD5 and endopiceasin., Results: Screening of a P. glauca EST database with sequences of known plant defensins identified four genes with homology to the known P. glauca defensin PgD1, which were designated PgD2-5. Whereas in the mature PgD2-4 only 7-9 amino acids differed from PgD1, PgD5 had only 64% sequence identity. PgD5 was amplified from P. glauca genomic DNA by PCR. It codes for a precursor of 77-amino acid that is fully conserved within the Picea genus and has similarity to plant defensins. Recombinant PgD5, produced in Escherichia coli, had a molecular mass of 5.721 kDa, as determined by mass spectrometry. The PgD5 peptide exhibited strong antifungal activity against several phytopathogens without any effect on the morphology of the treated fungal hyphae, but strongly inhibited hyphal elongation. A SYTOX uptake assay suggested that the inhibitory activity of PgD5 could be associated with altering the permeability of the fungal membranes. Another completely unrelated defensin gene was identified in the EST library and named endopiceasin. Its gene codes for a 6-cysteine peptide that shares high similarity with the fungal defensin plectasin., Conclusions: Screening of a P. glauca EST database resulted in the identification of five new defensin-encoding genes. PgD5 codes for a plant defensin that displays non-morphogenic antifungal activity against the phytopathogens tested, probably by altering membrane permeability. PgD5 has potential for application in the plant biotechnology sector. Endopiceasin appears to derive from an endo- or epiphytic fungal strain rather than from the plant itself.

Impacts of elevated ground-level ozone (O(3)) on nitrogen (N) uptake and allocation were studied on mature European beech (Fagus sylvatica L.) and Norway spruce (Picea abies [L.] Karst.) in a forest stand, hypothesizing that: (i) chronically elevated O(3) limits nutrient uptake, and (ii) beech responds more sensitively to elevated O(3) than spruce, as previously found for juvenile trees. Tree canopies were exposed to twice-ambient O(3) concentrations (2 × O(3)) by a free-air fumigation system, with trees under ambient O(3) serving as control. After 5 years of O(3) fumigation, (15)NH(4)(15)NO(3) was applied to soil, and concentrations of newly acquired N (N(labelled)) and total N (N(total)) in plant compartments and soil measured. Under 2 × O(3), N(labelled) and N(total) were increased in the bulk soil and tended to be lower in fine and coarse roots of both species across the soil horizons, supporting hypothesis (i). N(labelled) was reduced in beech foliage by up to 60%, and by up to 50% in buds under 2 × O(3). Similarly, N(labelled) in stem bark and phloem was reduced. No such reduction was observed in spruce, reflecting a stronger effect on N acquisition in beech in accordance with hypothesis (ii). In spruce, 2 × O(3) tended to favour allocation of new N to foliage. N(labelled) in beech foliage correlated with cumulative seasonal transpiration, indicating impaired N acquisition was probably caused by reduced stomatal conductance and, hence, water transport under elevated O(3). Stimulated fine root growth under 2 × O(3) with a possible increase of below-ground N sink strength may also have accounted for lowered N allocation to above-ground organs. Reduced N uptake and altered allocation may enhance the use of stored N for growth, possibly affecting long-term stand nutrition.

While downward photosynthetic acclimation in response to elevated CO(2) (EC) is frequently accompanied by reduction in Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), the exact mechanism behind this decrease and its dynamics are not well understood. We comprehensively studied Rubisco adjustment to EC in coniferous Picea abies using an electrophoretic (protein content), spectrophotometric (initial (RA(initial)) and total (RA(total)) in vitro Rubisco activities), and gas-exchange (maximum carboxylation activity in vivo (V(Cmax))) techniques. With respect to differing carbon sink strength and nitrogen remobilization, we hypothesized greater acclimation of photosynthesis in one-year-old as compared to current-year needles and at the end than at the beginning of the vegetation season. EC treatment led to a decrease in V(Cmax) values in current-year needles, but the ribulose-1,5-bisphosphate (RuBP)-limited rate of photosynthesis (J(max)) remained unaffected. Indeed, both V(Cmax) and J(max) were reduced by the EC treatment in one-year-old needles. The extent of photosynthetic acclimation in EC plants did not increase, however, during the vegetation season. EC decreased the activation state of Rubisco (RA(initial)/RA(total)) by 16% and 5% in current-year and one-year-old needles, respectively (averaged over the growing season). While during spring (short-term effect) EC treatment did not influence the Rubisco content per unit leaf area and decreased its specific activity (activity per unit Rubisco mass) in both needle age classes studied, exposure to EC during the entire vegetation season tended to reduce the Rubisco content while increasing its specific activity. Irrespective of CO(2) treatment and needle age, a hyperbolic-decay relationship was observed between Rubisco-specific activity and its content., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

To assess the effects of elevated CO(2) concentration ([CO(2)]) on the photosynthetic properties around spring budbreak, we monitored the total leaf sugar and starch content, and chlorophyll fluorescence in 1-year-old needles of Sakhalin spruce (Picea glehnii Masters) seedlings in relation to the timing of budbreak, grown in a phytotron under natural daylight at two [CO(2)] levels (ambient: 360 μmol mol(-1) and elevated: 720 μmol mol(-1)). Budbreak was accelerated by elevated [CO(2)] accompanied with earlier temporal declines in the quantum yield of PSII electron transport (Φ(PSII)) and photochemical quenching (q(L)). Plants grown under elevated [CO(2)] showed pre-budbreak leaf starch content twice as high with no significant difference in Φ(PSII) from ambient-CO(2)-grown plants when compared at the same measurement [CO(2)], i.e., 360 or 720 μmol mol(-1), suggesting that the enhanced pre-budbreak leaf starch accumulation might not cause down-regulation of photosynthesis in pre-existing needles under elevated [CO(2)]. Conversely, lower excitation pressure adjusted for the efficiency of PSII photochemistry ((1 - q(P)) F(v)'/F(m)') was observed in plants grown under elevated [CO(2)] around budbreak when compared at their growth [CO(2)] (i.e., comparing (1 - q(P)) F(v)'/F(m)' measured at 720 μmol mol(-1) in elevated-CO(2)-grown plants with that at 360 μmol mol(-1) in ambient-CO(2)-grown plants), which suggests lower rate of photoinactivation of PSII in the elevated-CO(2)-grown plants around spring budbreak. The degree of photoinhibition, as indicated by the overnight-dark-adapted F(v)/F(m), however, showed no difference between CO(2) treatments, thereby suggesting that photoprotection during the daytime or the repair of PSII at night was sufficient to alleviate differences in the rate of photoinactivation.

Norway spruce (Picea abies L. Karst) grown under ambient (365-377 μmol(CO(2)) mol(-1); AC) and elevated (700 μmol(CO(2)) mol(-1); EC) CO(2) concentrations within glass domes with automatically adjustable windows and on an open-air control site were studied after 8 years of treatment. The effect of EC on photosynthesis, mesophyll structure and phenolics accumulation in sun and shade needles was examined. Photosynthetic assimilation and dark respiration rates were measured gasometrically; the structural parameters of mesophyll were determined using confocal microscopy and stereological methods. The contents of total soluble phenolics and lignin were assessed spectrophotometrically, and localizations of different phenolic groups were detected histochemically on needle cross-sections. EC enhanced the light-saturated CO(2) assimilation rate and reduced dark respiration in the current-year needles. No effects of CO(2) enrichment on mesophyll structural parameters were observed. Similarly, the accumulation and localization of phenolics and lignin remained unaffected by EC treatment. Needles differentiated into sun and shade ecotypes in the same manner and to the same extent irrespective of CO(2) treatment. Based on these results, it is apparent that the EC-induced enhancement of photosynthesis is not related to changes in the examined structural parameters of mesophyll and accumulation of phenolic compounds., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Tree-ring width of Picea abies was studied along an altitudinal gradient in the Harz Mountains, Germany, in an area heavily affected by SO(2)-related forest decline in the second half of the 20th century. Spruce trees of exposed high-elevation forests had earlier been shown to have reduced radial growth at high atmospheric SO(2) levels. After the recent reduction of the SO(2) load due to clean air acts, we tested the hypothesis that stem growth recovered rapidly from the SO(2) impact. Our results from two formerly damaged high-elevation spruce stands support this hypothesis suggesting that the former SO(2)-related spruce decline was primarily due to foliar damage and not to soil acidification, as the deacidification of the (still acidic) soil would cause a slow growth response. Increasing temperatures and deposited N accumulated in the topsoil are likely additional growth-promoting factors of spruce at high elevations after the shortfall of SO(2) pollution., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The ozone effect on Norway spruce (Picea abies (L) Karst.) and European beech (Fagus sylvatica L.) was studied on 48 monitoring plots in 2005-2008. These plots represent two major forest tree species stands of different ages in eight regions of the Czech Republic. The forest conditions were represented by defoliation and the annual radial increment of individual trees. The ozone exposure was assessed by using modeled values of mean annual O(3) concentration and the AOT40 index. The malondialdehyde (MDA) content of the foliage was analysed and used as an indicator of oxidative stress. The correlation analysis showed a significant relation of Norway spruce defoliation to the AOT40 exposure index, and European beech defoliation to the MDA level. The radial increment response to ozone was significant only for the European beech: (a) the correlation analysis showed its decrease with increasing AOT40; (b) the regression model showed its decrease with increasing mean annual ozone concentration only at lower altitudes (<700 m a.s.l.).

The ability to assess the toxic potential of soil contamination within boreal regions is currently limited to test species representative of arable lands. This study evaluated the use of six boreal plant species (Pinus banksiana, Picea glauca, Picea mariana, Populus tremuloides, Calamagrostis Canadensis, and Solidago canadensis) and four invertebrate species (Dendrodrilus rubidus, Folsomia nivalis, Proisotoma minuta, and Oppia nitens) and compared their performance to a suite of standard agronomic soil test species using site soils impacted by petroleum hydrocarbon (PHC) and salt contamination. To maintain horizon-specific differences, individual soil horizons were collected from impacted sites and relayered within the test vessels. Use of the boreal species was directly applicable to the assessment of the contaminated forest soils and, in the case of the hydrocarbon-impacted soil, demonstrated greater overall sensitivity (25th percentile of estimated species sensitivity distribution [ESSD25] = 5.6% contamination: 10,600 mg/kg fraction 3 [F3; equivalent hydrocarbon range of >C16 to C34] Of/Oh horizon, and 270 mg/kg F3 Ahg horizon) relative to the standard test species (ESSD25 = 23% contamination: 44,000 mg/kg F3 Of/Oh horizon, and 1,100 mg/kg F3 Ahg horizon). For salinity, there was no difference between boreal and standard species with a combined ESSD25 = 2.3%, equating to 0.24 and 0.25 dS/m for the Ah and Ck horizons. The unequal distribution of soil invertebrates within the layered test vessels can confound test results and the interpretation of the toxic potential of a site. The use of test species relevant to boreal eco-zones strengthens the applicability of the data in support of realistic ecological risk assessments applicable to the boreal regions., (Copyright © 2012 SETAC.)

Somatic embryogenesis is used for vegetative propagation of conifers. Embryogenic cultures can be established from zygotic embryos; however, the embryogenic potential decreases during germination. In Arabidopsis, LEAFY COTYLEDON (LEC) genes are expressed during the embryonic stage, and must be repressed to allow germination. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) causes de-repression of LEC genes. ABSCISIC ACID3 (ABI3) and its Zea mays ortholog VIVIPAROUS1 (VP1) act together with the LEC genes to promote embryo maturation. In this study, we have asked the question whether TSA treatment in a conifer affects the embryogenic potential and the expression of embryogenesis-related genes. We isolated two conifer LEC1-type HAP3 genes, HAP3A and HAP3B, from Picea abies and Pinus sylvestris. A comparative phylogenetic analysis of plant HAP3 genes suggests that HAP3A and HAP3B are paralogous genes originating from a duplication event in the conifer lineage. The expression of HAP3A is high, in both somatic and zygotic embryos, during early embryo development, but decreases during late embryogeny. In contrast, the expression of VP1 is initially low but increases during late embryogeny. After exposure to TSA, germinating somatic embryos of P. abies maintain the competence to differentiate embryogenic tissue, and simultaneously the germination progression is partially inhibited. Furthermore, when embryogenic cultures of P. abies are exposed to TSA during embryo maturation, the maturation process is arrested and the expression levels of PaHAP3A and PaVP1 are maintained, suggesting a possible link between chromatin structure and expression of embryogenesis-related genes in conifers.

Meta-analyses reveal that fast-growing species have a greater growth response to elevated CO(2) than slow-growing species. It is unknown whether this is a direct response or whether inter-specific differences in growth are simply correlated with other physiological or morphological differences among species that affect the growth response to CO(2). Here we use intra-specific variation in Picea glauca to examine the mechanistic basis for this relationship. Relative growth rate (RGR) of 29 genotypes grown at ambient (370 µl l(-1)) or elevated (740 µl 1(-1)) CO(2) was measured. Physiological and morphological traits describing differences in allocation, canopy structure, stomatal function and photosynthesis were determined. Most variation in RGR (74%) was explained by traits associated with canopy structure. Although there was a strong correlation between RGR(740) and RGR(370), we found no evidence that genotypes that grew fast at ambient CO(2) had a greater relative growth response to CO(2). Given that the pattern found at the intra-specific level differed from that reported at the inter-specific level, our results suggest that RGR per se does not affect the growth response to CO(2). Rather, the CO(2) growth response is determined by traits that may or may not be correlated with RGR.

A comparable series of specimens from spruce wood were pre-treated with sodium hydroxide, sodium hydroxide and hydrogen peroxide, or per-acetic acid sequences. The pre-treatments reduced the yield of pulps and their Kappa number noticeably, diminished the degree of polymerization moderately, and increased their brightness. One-step peroxide bleaching of pulps from the pre-treated spruce wood resulted in their higher brightness compared to bleached pulp from sound wood. From the viewpoint of improved properties of pulp, the most efficient were the sodium hydroxide/per-acetic acid and per-acetic acid/sodium hydroxide sequences. The pre-treatments did not influence mechanical strength of the obtained pulps significantly., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

By employing metal salts in dilute-acid pretreatment the severity can be reduced due to reduced activation energy. This study reports on a dilute-acid steam pretreatment of spruce chips by addition of a small amount of ferrous sulfate to the acid catalyst, i.e., either SO2, H2SO3 or H2SO4. The utilization of ferrous sulfate resulted in a slightly increased overall glucose yield (from 74% to 78% of the theoretical value) in pretreatment with SO2 and H2SO3. Impregnation with ferrous sulfate and sulfuric acid did not give any improvement compared with pretreatment based solely on H2SO4., (Copyright © 2010. Published by Elsevier Ltd.)

Inhibitory lignocellulose hydrolysates were treated with the reducing agents dithionite and sulfite to achieve improved fermentability. Addition of these reducing agents (in the concentration range 5.0-17.5 mM) to enzymatic hydrolysates of spruce wood or sugarcane bagasse improved processes based on both SHF (simultaneous hydrolysis and fermentation) and SSF (simultaneous saccharification and fermentation). The approach was exemplified in ethanolic fermentations with Saccharomyces cerevisiae and by using hydrolysates with sugar concentrations>100 g/L (for SHF) and with 10% dry-matter content (for SSF). In the SHF experiments, treatments with dithionite raised the ethanol productivities of the spruce hydrolysate from 0.2 to 2.5 g×L(-1)×h(-1) and of the bagasse hydrolysate from 0.9 to 3.9 g×L(-1)×h(-1), values even higher than those of fermentations with reference sugar solutions without inhibitors. Benefits of the approach include that the addition of the reducing agent can be made in-situ directly in the fermentation vessel, that the treatment can be performed at a temperature and pH suitable for fermentation, and that the treatment results in dramatically improved fermentability without degradation of fermentable sugars. The many benefits and the simplicity of the approach offer a new way to achieve more efficient manufacture of fermentation products from lignocellulose hydrolysates., (Copyright © 2010 Elsevier Ltd. All rights reserved.)