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• In seed plants, current knowledge concerning embryonic pattern formation by polar auxin transport (PAT) and WUSCHEL-related homeobox (WOX) gene activity is primarily derived from studies on angiosperms, while less is known about these processes in gymnosperms. In view of the differences in their embryogeny, and the fact that somatic embryogenesis is used for mass propagation of conifers, a better understanding of embryo development is vital. • The expression patterns of PaWOX2 and PaWOX8/9 were followed with quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH) during seed and somatic embryo development in Norway spruce (Picea abies), and in somatic embryos treated with the PAT inhibitor N-1-naphthylphthalamic acid (NPA). • Both PaWOX2 and PaWOX8/9 were highly expressed at the early growth stages of zygotic and somatic embryos, and shared a similar expression pattern over the entire embryo. At later embryo stages, high expression of PaWOX8/9 became restricted to cotyledon primordia, epidermis, procambium and root apical meristem (RAM), which became most evident in NPA-treated somatic embryos, while expression of PaWOX2 was much lower. • Our results suggest an ancestral role of WOX in seed plant embryo development, and strengthen the proposed connection between PAT, PIN-FORMED (PIN) and WOX in the regulation of embryo patterning in seed plants.

Canopies of adult European beech (Fagus sylvatica) and Norway spruce (Picea abies) were labeled with CO(2) depleted in (13)C to evaluate carbon allocation belowground. One-half the trees were exposed to elevated O(3) for 6 yrs prior to and during the experiment. Soil-gas sampling wells were placed at 8 and 15 cm and soil CO(2) was sampled during labeling in mid-late August, 2006. In beech, delta(13)CO(2) at both depths decreased approximately 50 h after labeling, reflecting rapid translocation of fixed C to roots and release through respiration. In spruce, label was detected in fine-root tissue, but there was no evidence of label in delta(13)CO(2). The results show that C fixed in the canopy rapidly reaches respiratory pools in beech roots, and suggest that spruce may allocate very little of recently-fixed carbon into root respiration during late summer. A change in carbon allocation belowground due to long-term O(3) exposure was not observed.

Malondialdehyde (MDA), a product of lipid peroxidation and biomarker of oxidative stress, is measured over the long term in spruce Picea abies needles under real conditions in three Czech mountain border areas. The trends presented collate the MDA content in spruce needles with ambient ozone, temperature and precipitation as casual, and defoliation as a subsequent factor for the period 1994-2006. We have found the overall decreasing trends in MDA and defoliation. The highest MDA and defoliation are recorded in the Jizerske, the lowest in the Krusne hory Mts. Out of the examined variables the MDA is predicted best by mean temperature in vegetation season, median of O(3) concentrations and AOT40; these three variables account for 34% of MDA1 and 36% of MDA2 variability. Our hypothesis that higher ambient O(3) exposure results in higher MDA contents in P. abies needles under real conditions has not been approved., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Synthesis of 5-aminolevulinic acid (ALA) represents a rate limiting step in the tetrapyrrole biosynthetic pathway, and is regulated by metabolic feedback control of glutamyl-tRNA reductase (GluTR) activity. The FLU protein has been attributed to this regulation. Later in the biosynthetic pathway, reduction of protochlorophyllide (Pchlide), catalyzed by protochlorophyllide oxidoreductase (POR), ensures another important regulatory step in the chlorophyll biosynthesis. In the present work, we investigated the expression and cellular abundance of nuclear-encoded and plastid-encoded proteins involved in ALA synthesis and Pchlide reduction in Norway spruce (Picea abies L. Karst.) as a representative of plant species with high ability to synthesize chlorophyll in the dark. Using dark-grown, light/dark-grown and gabaculine-treated seedlings, we demonstrated that gabaculine-impaired etiochloroplast and chloroplast development has no negative effect on GluTR accumulation in the cotyledons. However, in contrast to control plants, the relative amount of GluTR was similar both in the dark-grown and light/dark-grown gabaculine-treated seedlings. We identified a partial sequence of the FLU-like gene in Norway spruce, and using antibodies against the FLU-like protein (FLP), we showed that FLP accumulated mostly in the dark-grown control seedlings and gabaculine-treated seedlings. In contrast to nuclear-encoded GluTR and FLP, accumulation of plastid-encoded light-independent POR (DPOR) was sensitive to gabaculine treatment. The levels of DPOR subunits were substantially lower in the light/dark-grown control seedlings and gabaculine-treated seedlings, although the corresponding genes chlL, chlN and chlB were expressed. Since we analyzed the samples with different plastid types, plastid ultrastructure and physiological parameters like Pchlide and chlorophyll contents, in vivo chlorophyll fluorescence and photosynthetic efficiency of the seedlings were characterized. Apart from etiochloroplast-specific accumulation of the DPOR subunits, we described, in some detail, additional specific features of chlorophyll biosynthesis in the spruce seedlings that differ from those known in angiosperms., (Copyright 2010 Elsevier GmbH. All rights reserved.)

Stomatal O3 fluxes to a mixed beech/spruce stand (Fagus sylvatica/Picea abies) in Central Europe were determined using two different approaches. The sap flow technique yielded the tree-level transpiration, whereas the eddy covariance method provided the stand-level evapotranspiration. Both data were then converted into stomatal ozone fluxes, exemplifying this novel concept for July 2007. Sap flow-based stomatal O3 flux was 33% of the total O3 flux, whereas derivation from evapotranspiration rates in combination with the Penman-Monteith algorithm amounted to 47%. In addition to this proportional difference, the sap flow-based assessment yielded lower levels of stomatal O3 flux and reflected stomatal regulation rather than O3 exposure, paralleling the daily courses of canopy conductance for water vapor and eddy covariance-based total stand-level O3 flux. The demonstrated combination of sap flow and eddy covariance approaches supports the development of O3 risk assessment in forests from O3 exposure towards flux-based concepts., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The performance of two pretreatment methods, sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) and dilute acid (DA), was compared in pretreating softwood (spruce) for fuel ethanol production at 180 degrees Celsius for 30 min with a sulfuric acid loading of 5% on oven-dry wood and a 5:1 liquor-to-wood ratio. SPORL was supplemented with 9% sodium sulfite (w/w of wood). The recoveries of total saccharides (hexoses and pentoses) were 87.9% (SPORL) and 56.7% (DA), while those of cellulose were 92.5% (SPORL) and 77.7% (DA). The total of known inhibitors (furfural, 5-hydroxymethylfurfural, and formic, acetic and levulinic acids) formed in SPORL were only 35% of those formed in DA pretreatment. SPORL pretreatment dissolved approximately 32% of the lignin as lignosulfonate, which is a potential high-value co-product. With an enzyme loading of 15 FPU (filter paper units) per gram of cellulose, the cellulose-to-glucose conversion yields were 91% at 24h for the SPORL substrate and 55% at 48 h for the DA substrate, respectively., (Copyright 2009 Elsevier Ltd. All rights reserved.)

In a 50- to 70-year-old mixed stand of Norway spruce (Picea abies (L.) Karst.) and European beech (Fagus sylvatica L.) in Germany, tree cohorts have been exposed to double ambient ozone (2xO(3)) from 2000 through 2007 and can be compared with trees in the same stand under the ambient ozone regime (1xO(3)). Annual diameter growth, allocation pattern, stem form, and stem volume were quantified at the individual tree and stand level. Ozone fumigation induced a shift in the resource allocation into height growth at the expense of diameter growth. This change in allometry leads to rather cone-shaped stem forms and reduced stem stability in the case of spruce, and even neiloidal stem shapes in the case of beech. Neglect of such ozone-induced changes in stem shape may lead to a flawed estimation of volume growth. On the stand level, 2xO(3) caused, on average, a decrease of 10.2 m(3) ha(-1) yr(-1) in European beech., (2009 Elsevier Ltd. All rights reserved.)

The effects of experimentally elevated O(3) on soil respiration rates, standing fine-root biomass, fine-root production and delta(13)C signature of newly produced fine roots were investigated in an adult European beech/Norway spruce forest in Germany during two subsequent years with contrasting rainfall patterns. During humid 2002, soil respiration rate was enhanced under elevated O(3) under beech and spruce, and was related to O(3)-stimulated fine-root production only in beech. During dry 2003, the stimulating effect of O(3) on soil respiration rate vanished under spruce, which was correlated with decreased fine-root production in spruce under drought, irrespective of the O(3) regime. delta(13)C signature of newly formed fine-roots was consistent with the differing g(s) of beech and spruce, and indicated stomatal limitation by O(3) in beech and by drought in spruce. Our study showed that drought can override the stimulating O(3) effects on fine-root dynamics and soil respiration in mature beech and spruce forests., (2009 Elsevier Ltd. All rights reserved.)

Here we synthesize key findings from a series of experiments to gain new insight on inter-plant competition between juvenile beech (Fagus sylvatica) and spruce (Picea abies) under the influence of increased O(3) and CO(2) concentrations. Competitiveness of plants was quantified and mechanistically interpreted as space-related resource investments and gains. Stable isotopes were addressed as temporal integrators of plant performance, such as photosynthesis and its relation to water use and nitrogen uptake. In the weaker competitor, beech, efficiency in space-related aboveground resource investment was decreased in competition with spruce and positively related to Delta(13)C, as well as stomatal conductance, but negatively related to delta(18)O. Likewise, our synthesis revealed that strong belowground competition for water in spruce was paralleled in this species by high N assimilation capacity. We suggest combining the time-integrative potential of stable isotopes with space-related investigations of competitiveness to accomplish mechanistic understanding of plant competition for resources., (2009 Elsevier Ltd. All rights reserved.)

Salmine, an arginine-rich protamine, is explored for its concentration-dependent potential to restructure the genome and remodel the homeotic development of Norway spruce embryos expressing monozygotic cleavage polyembryony (MCP). In controls and at low salmine, two protein fractions on SDS-PAGE gels were associated with cells responsible for generating the basal plan for early embryogenesis. With high salmine, embryonal initials no longer differentiated into embryonal tubes. Embryos having embryonal tubes no longer enucleated and differentiated into embryonal suspensors. Biomass and amino acid N declined. Nuclear and cytoplasmic organization was disrupted and nucleoli were highly vacuolated. The transcription of the two protein fractions, PCNA (cyclin) activity and MCP were blocked. Cellular proteins were turned over by proteasomal ubiquitination and others released into the culture medium. Biomass loss and gluconeogenesis of amino acids led to the accumulation of free arginine N. No evidence was obtained with salmine for the remodeling of cells into gametes.

Strategic control of mitochondrial movements and cellular distribution is essential for correct cell function and survival. However, despite being a vital process, mitochondrial movement in plant cells is a poorly documented phenomenon. To investigate the roles of actin filaments and microtubules on mitochondrial movements, Picea wilsonii pollen tubes were treated with two microtubule-disrupting drugs, two actin-disrupting drugs and a myosin inhibitor. Following these treatments, mitochondrial movements were characterized by multiangle evanescent wave microscopy and laser-scanning confocal microscopy. The results showed that individual mitochondria underwent three classes of linear movement: high-speed movement (instantaneous velocities >5.0 microm/s), low-speed movement (instantaneous velocities <5.0 microm/s) and variable-speed movement (instantaneous velocities ranging from 0.16 to 10.35 microm/s). 10 nM latrunculin B induced fragmentation of actin filaments and completely inhibited mitochondrial vectorial movement. Jasplakinolide treatment induced a 28% reduction in chondriome motility, and dramatically inhibition of high-speed and variable-speed movements. Treatment with 2,3-butanedione 2-monoxime caused a 61% reduction of chondriome motility, and the complete inhibition of high-speed and low-speed movements. In contrast to actin-disrupting drugs, microtubule-disrupting drugs caused mild effects on mitochondrial movement. Taxol increased the speed of mitochondrial movement in cortical cytoplasm. Oryzalin induced curved mitochondrial trajectories with similar velocities as in the control pollen tubes. These results suggest that mitochondrial movement at low speeds in pollen tubes is driven by myosin, while high-speed and variable-speed movements are powered both by actin filament dynamics and myosin. In addition, microtubule dynamics has profound effects on mitochondrial velocity, trajectory and positioning via its role in directing the arrangement of actin filaments.

The impact of elevated ozone concentrations on early ontogenetic stages of pine (Pinus sylvestris) and spruce (Picea abies, Picea obovata, P. abies x P. obovata) seedlings originating from different provenances in Russia were studied in the open-field ozone fumigation system located in Kuopio, Finland, over a span of 2 y. The AOT40 value (accumulated ozone dose over the threshold 40 ppb during daylight hours) was 11 ppm hr per growing season, which was 1.4 times higher than the ambient air concentration. The plants were measured for germination rate; shoot increment; needle length; and dry mass of needles, shoots, and roots. Significant differences between pine and spruce provenance response to ozone were found in all parameters. Ozone stress immediately reduced the germination rate of Northern pine provenances, whereas biomass reductions became evident during the second year of the exposure in all pine provenances. Spruce species were more tolerant to elevated ozone concentrations. Our results indicate that seedling development is vulnerable to increasing ozone concentrations and that attention must be paid to the provenance selection.

It has been suggested that volatile organic compounds (VOCs) are involved in organic aerosol formation, which in turn affects radiative forcing and climate. The most abundant VOCs emitted by terrestrial vegetation are isoprene and its derivatives, such as monoterpenes and sesquiterpenes. New particle formation in boreal regions is related to monoterpene emissions and causes an estimated negative radiative forcing of about -0.2 to -0.9 W m(-2). The annual variation in aerosol growth rates during particle nucleation events correlates with the seasonality of monoterpene emissions of the local vegetation, with a maximum during summer. The frequency of nucleation events peaks, however, in spring and autumn. Here we present evidence from simulation experiments conducted in a plant chamber that isoprene can significantly inhibit new particle formation. The process leading to the observed decrease in particle number concentration is linked to the high reactivity of isoprene with the hydroxyl radical (OH). The suppression is stronger with higher concentrations of isoprene, but with little dependence on the specific VOC mixture emitted by trees. A parameterization of the observed suppression factor as a function of isoprene concentration suggests that the number of new particles produced depends on the OH concentration and VOCs involved in the production of new particles undergo three to four steps of oxidation by OH. Our measurements simulate conditions that are typical for forested regions and may explain the observed seasonality in the frequency of aerosol nucleation events, with a lower number of nucleation events during summer compared to autumn and spring. Biogenic emissions of isoprene are controlled by temperature and light, and if the relative isoprene abundance of biogenic VOC emissions increases in response to climate change or land use change, the new particle formation potential may decrease, thus damping the aerosol negative radiative forcing effect.

Hydrocarbon-contaminated wastes generated from oil and gas drilling activities may be used as a soil amendment once composted and further decomposition of residual hydrocarbons can be accomplished after the composts are applied to soils. To test if N fertilization may enhance hydrocarbon decomposition, we investigated the effects of N application on hydrocarbon degradation in different-aged composts (1-, 2-, 3-, and 4-year-old composts, coded as 1Y, 2Y, 3Y, and 4Y composts, respectively) through a pot experiment planted with white spruce (Picea glauca [Moench] Voss) seedlings. The percentage degradation of total petroleum hydrocarbon (TPH, C11 to C40) in the composts without N fertilization was correlated to initial NH4+ concentrations (R = 0.99, P < 0.001). The percentage degradation of TPH was highest in the 3Y compost (41.1%) that had an initial level of 325.3 mg NH4+ -N kg(-1) and the lowest in the IY compost (9.3%) that had an initial level of 8.3 mg NH4+ -N kg(-1). The degradation of TPH was enhanced by Nfertilization in the 1Y (from 9.3 to 15.3%) and 4Y composts (from 14.3 to 22.6%) that had low initial NH4+ concentrations. Our results show that application of NH4+ -based fertilizers may enhance the degradation of TPH when initial NH4+ concentrations in the compost are low.

We examined the effect of ozone (O(3)) on Norway spruce (Picea abies) needle epicuticular wax over three seasons at the Kranzberg Ozone Fumigation Experiment. Exposure to 2x ambient O(3) ranged from 64.5 to 74.2 microl O(3) l(-1) h AOT40, and 117.1 to 123.2 nl O(3) l(-1) 4th highest daily maximum 8-h average O(3) concentration. The proportion of current-year needle surface covered by wax tubes, tube aggregates, and plates decreased (P=0.011) under 2x O(3). Epistomatal chambers had increased deposits of amorphous wax. Proportion of secondary alcohols varied due to year (P=0.004) and O(3) treatment (P=0.029). Secondary alcohols were reduced by 9.1% under 2x O(3). Exposure to 2x O(3) increased (P=0.037) proportions of fatty acids by 29%. Opposing trends in secondary alcohols and fatty acids indicate a direct action of O(3) on wax biosynthesis. These results demonstrate O(3)-induced changes in biologically important needle surface characteristics of 50-year-old field-grown trees.

White spruce is an important commercial species for reforestation. The success in its propagation through somatic embryogenesis is well documented; however the physiological processes involved are poorly understood and remain unoptimized. The variable quality embryos generated in vitro from the same genotype suggest control at the protein and metabolite level. In order to probe metabolic changes, we have conducted a "metabolic footprinting" study, whereby culture media from growing cells was quantitatively analyzed to determine which metabolites were consumed and excreted. Such experiments are advantageous in that there is no need to quench cellular metabolism or extract intracellular metabolites through time-consuming protocols. In this paper we demonstrate the application of the footprinting assay to somatic embryo cells of white spruce (Picea glauca) using 1D (1)H NMR spectroscopy. We have surveyed embryogenesis metabolism in two types of media, maintenance (MN) and maturation (MT). MN medium does not result in shoot apical meristem (SAM) formation, while MT medium induces the necessary changes leading to fully developed somatic embryos. The two types of media were easily distinguished using metabolomics analysis, namely multivariate pattern recognition statistics (orthogonal partial least squares discriminatory analysis). From this analysis, we have identified numerous compounds involved with branched chain amino acid pathways such as valine and isoleucine. These results are explained on the basis of known metabolic pathways implicated in plant and animal developmental processes, and ultimately implicate altered CoA biosynthesis.

This study established a novel process using sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) for robust and efficient bioconversion of softwoods. The process consists of sulfite treatment of wood chips under acidic conditions followed by mechanical size reduction using disk refining. The results indicated that after the SPORL pretreatment of spruce chips with 8-10% bisulfite and 1.8-3.7% sulfuric acid on oven dry (od) wood at 180 degrees C for 30 min, more than 90% cellulose conversion of substrate was achieved with enzyme loading of about 14.6 FPU cellulase plus 22.5 CBU beta-glucosidase per gram of od substrate after 48 h hydrolysis. Glucose yield from enzymatic hydrolysis of the substrate per 100 g of untreated od spruce wood (glucan content 43%) was about 37 g (excluding the dissolved glucose during pretreatment). Hemicellulose removal was found to be as critical as lignin sulfonation for cellulose conversion in the SPORL process. Pretreatment altered the wood chips, which reduced electric energy consumption for size reduction to about 19 Wh/kg od untreated wood, or about 19 g glucose/Wh electricity. Furthermore, the SPORL produced low amounts of fermentation inhibitors, hydroxymethyl furfural (HMF) and furfural, of about 5 and 1 mg/g of untreated od wood, respectively. In addition, similar results were achieved when the SPORL was applied to red pine. By building on the mature sulfite pulping and disk refining technologies already practiced in the pulp and paper industry, the SPORL has very few technological barriers and risks for commercialization.

Effects of ambient and elevated temperature and atmospheric carbon dioxide concentration ([CO2]) on CO2 assimilation rate and the structural and phenological development of shoots during their first growing season were studied in 45-year-old Norway spruce trees (Picea abies (L.) Karst.) enclosed in whole-tree chambers. Continuous measurements of net assimilation rate (NAR) in individual buds and shoots were made from early bud development to late August in two consecutive years. The largest effect of elevated temperature (TE) was manifest early in the season as an earlier start and completion of shoot length development, and a 1-3-week earlier shift from negative to positive NAR compared with the ambient temperature (TA) treatments. The largest effect of elevated [CO2] (CE) was found later in the season, with a 30% increase in maximum NAR compared with trees in the ambient [CO2] treatments (CA), and shoots assimilating their own mass in terms of carbon earlier in the CE treatments than in the CA treatments. Once the net carbon assimilation compensation point (NACP) had been reached, TE had little or no effect on the development of NAR performance, whereas CE had little effect before the NACP. No interactive effects of TE and CE on NAR were found. We conclude that in a climate predicted for northern Sweden in 2100, current-year shoots of P. abies will assimilate their own mass in terms of carbon 20-30 days earlier compared with the current climate, and thereby significantly contribute to canopy assimilation during their first year.

Auxin and polar auxin transport have been implicated in controlling embryo patterning and development in angiosperms but less is known from the gymnosperms. The aims of this study were to determine at what stages of conifer embryo development auxin and polar auxin transport are the most important for normal development and to analyze the changes in embryos after treatment with the polar auxin inhibitor N-1-naphthylphthalamic acid (NPA). For these studies, somatic embryos of Norway spruce (Picea abies L. Karst) were used. Growth on medium containing NPA leads to the formation of embryos with poor shoot apical meristem (SAM) and fused cotyledons, and to a pin-formed phenotype of the regenerated plantlets. The effect of NPA on embryo morphology was most severe if embryos were transferred to NPA-containing medium immediately before cotyledon initiation and SAM specification. Indole-3-acetic acid (IAA) was identified by immunolocalization in developing embryos. The highest staining intensity was seen in early staged embryos and then decreased as the embryos matured. No clear IAA-maxima was seen, although the apical parts of embryos, particularly the protoderm, and the suspensor cells appear to accumulate more IAA, as reflected by the staining pattern. The NPA treatment also caused expanded procambium and a broader root apical meristem in embryos, and a significant increase in the expression of a PIN1-like gene. Taken together, our results show that, for proper cotyledon initiation, correct auxin transport is needed only during a short period at the transition stage of embryo development, probably involving PIN efflux proteins and that a common mechanism is behind proper cotyledon formation within the species of angiosperms and conifers, despite their cotyledon number which normally differs.

Ca2+-calmodulin (Ca2+-CaM) is a critical molecule that mediates cellular functions by interacting with various metabolic and signaling pathways. However, the protein expression patterns and accompanying serial cytological responses in Ca2+-CaM signaling deficiency remain enigmatic. Here, we provide a global analysis of the cytological responses and significant alterations in protein expression profiles after trifluoperazine treatment in Picea meyeri, which abrogates Ca2+-CaM signaling. Ninety-three differentially displayed proteins were identified by comparative proteomics at different development stages and were assigned to different functional categories closely related to tip growth machinery. The inhibition of Ca2+-CaM signaling rapidly induced an increase in extracellular Ca2+ influx, resulting in dramatically increased cytosolic Ca2+ concentrations and ultrastructural abnormalities in organelles as the primary responses. Secondary and tertiary alterations included actin filament depolymerization, disrupted patterns of endocytosis and exocytosis, and cell wall remodeling, ultimately resulting in perturbed pollen tube extension. In parallel with these cytological events, time-course experiments revealed that most differentially expressed proteins showed time-dependent quantitative changes (i.e. some signaling proteins and proteins involved in organelle functions and energy production changed first, followed by alterations in proteins related to cytoskeletal organization, secretory pathways, and polysaccharide synthesis). Taken together, Ca2+-CaM dysfunction induced serial cytological responses and temporal changes in protein expression profiles, indicating the pivotal role of Ca2+-CaM in the regulation of tip growth machinery.

Black spruce (Picea mariana), white spruce (Picea glauca) and jack pine (Pinus banksiana) were inoculated with Suillus tomentosus and subjected to potassium fluoride (1 mM KF and 5 mM KF) in the presence and absence of 60 mM NaCl. The NaCl and KF treatments reduced total dry weights in jack pine and black spruce seedlings, but they did not affect total dry weights in white spruce seedlings. The addition of 60 mM NaCl to KF treatment solutions alleviated fluoride-induced needle injury in ectomycorrhizal (ECM) black spruce and white spruce, but had little effect in jack pine seedlings. Both KF and 60 mM NaCl treatments reduced E values compared with non-treated control seedlings. However, with the exception of small reductions of K(r) by NaCl treatments in black spruce, the applied KF and NaCl treatments had little effect on K(r) in ECM plants. Chloride tissue concentrations in NaCl-treated plants were not affected by the presence of KF in treatment solutions. However, shoot F concentrations in ECM black spruce and white spruce treated with 5 mM KF + 60 mM NaCl were significantly reduced compared with the 5 mM KF treatment. The results point to a possible competitive inhibition of F transport by Cl. We also suggest that the possibility that aquaporins may be involved in the transmembrane transport of F should be further investigated.

Defence in young trees has been much less studied than defence in older ones. In conifers, resin within ducts in bark is an important quantitative defence, but its expression in young trees may be influenced by developmental or physical constraints on the absolute size of the resin ducts as well as by differential allocation of resources to growth and resin synthesis. To examine these relationships, we used nitrogen fertilisation of 1- and 2-year-old pine and spruce to produce trees of different sizes and measured the effect on the number and size of resin ducts and the amount of resin they contained. All of these variables tended to increase with stem diameter, indicating a positive relationship between resin-based defence and growth of 1- and 2-year-old trees. In pine, however, the mass of resin flowing from severed ducts was much lower relative to duct area in 1- than in 2-year-old trees, suggesting that the older trees allocated a higher proportion of the carbon budget to resin synthesis. Resin-based defence in 1-year-old pines appears to be both positively related to growth and resource limited. In spruce, resin production was generally lower, and age-related differences were not observed, suggesting that resin-based defence is less important in this species. Bio-assays of 2-year-old trees with the pine weevil, Hylobius abietis, emphasised the importance of resin as a defence against this bark feeding insect. Nitrogen fertilisation had a limited influence on resistance expression. One-year-old trees remained susceptible because of their small size, low resin production and limited response to fertilisation. The strong growth response of 2-year-old trees to fertilisation increased resin-based defence, but most spruce trees remained susceptible, while most pines were resistant at all levels of fertilisation.

We analyzed effects of nitrogen availability and form on growth rates, concentrations of polyamines and inorganic ions and glutamine synthetase activity in in-vitro-cultured red spruce (Picea rubens Sarg.) cells. Growth rates, concentrations of polyamines and glutamine synthetase activity declined when either the amount of nitrate or the total amount of N in the culture medium was reduced. When total N in the medium was increased, cell mass increased without significant changes in glutamine synthetase activity or polyamine concentration. Reductions in the amount of nitrate or total N in the culture medium resulted in increased accumulations of Ca, Mn and Zn in the cells, and K accumulation decreased in response to decreasing nitrate:ammonium ratios. The data indicate that changes in total N availability as well as the forms of N play important roles in the physiological responses of in-vitro-grown red spruce cells that mimic the observed responses of forest trees to soil N deficiency and N fertilization.

In fall (November 2005) and winter (February 2006), we collected current-year foliage of native red spruce (Picea rubens Sarg.) growing in a reference watershed and in a watershed treated in 1999 with wollastonite (CaSiO(3), a slow-release calcium source) to simulate preindustrial soil calcium concentrations (Ca-addition watershed) at the Hubbard Brook Experimental Forest (Thornton, NH). We analyzed nutrition, soluble sugar concentrations, ascorbate peroxidase (APX) activity and cold tolerance, to evaluate the basis of recent (2003) differences between watersheds in red spruce foliar winter injury. Foliar Ca and total sugar concentrations were significantly higher in trees in the Ca-addition watershed than in trees in the reference watershed during both fall (P=0.037 and 0.035, respectively) and winter (P=0.055 and 0.036, respectively). The Ca-addition treatment significantly increased foliar fructose and glucose concentrations in November (P=0.013 and 0.007, respectively) and foliar sucrose concentrations in winter (P=0.040). Foliar APX activity was similar in trees in both watersheds during fall (P=0.28), but higher in trees in the Ca-addition watershed during winter (P=0.063). Cold tolerance of foliage was significantly greater in trees in the Ca-addition watershed than in trees in the reference watershed (P<0.001). Our results suggest that low foliar sugar concentrations and APX activity, and reduced cold tolerance in trees in the reference watershed contributed to their high vulnerability to winter injury in 2003. Because the reference watershed reflects forest conditions in the region, the consequences of impaired physiological function caused by soil Ca depletion may have widespread implications for forest health.

Maturation of Norway spruce (Picea abies L.) somatic embryos is induced by abscisic acid (ABA). Several proteins were proven to be involved in ABA sensing including ABI3/VP1 transcription factors and their orthologue PaVP1 was characterized in spruce. To evaluate the role of PaVP1 both in embryogenic potential and in the process of embryo maturation, we studied PaVP1 expression in lines with contrast embryogenic capacities in parallel with detailed anatomical characterization. PaVP1 expression was determined by northern blot hybridisation, which revealed presence of two differentially regulated VP1-like B3-domain transcripts. Full-length PaVP1 transcript level was negligible in all lines on the proliferation media, but it differed strongly on the maturation media containing ABA. In non-embryogenic line, lacking any differentiated structures, the transcript remained undetectable. In contrast, in embryogenic lines with meristematic centres attached to suspensor cells, PaVP1 expression increased strongly after transition onto the maturation media. In highly embryogenic lines, it kept on a high level until the embryos reached cotyledonary stage, while in developmentally arrested line incapable to form mature embryos, the expression dropped down in connection with advanced disintegration of the meristematic centres. Removal of ABA from the maturation media after 2 weeks of maturation resulted in aberrant embryo development and rapid decrease in PaVP1 expression, indicating the impact of exogenously supplemented ABA on both initiation and maintenance of PaVP1 expression and proper embryo development. Since permanently high or increasing PaVP1 transcript levels accompanied proper embryo development in all experiments, it could be regarded as a good marker of this process.

Two potent drugs, neomycin and TMB-8, which can block intracellular calcium release, were used to investigate their influence on pollen tube growth and cell wall deposition in Picea wilsonii. Apart from inhibiting pollen germination and pollen tube growth, the two drugs largely influenced tube morphology. The drugs not only obviously disturbed the generation and maintenance of the tip-localized Ca(2+) gradient but also led to a heavy accumulation of callose at the tip region of P. wilsonii pollen tubes. Fourier transform infrared (FTIR) spectroscopy analysis showed that the deposition of cell wall components, such as carboxylic acid, pectins, and other polysaccharides, in pollen tubes was changed by the two drugs. The results obtained from immunolabeling with different pectin and arabinogalactan protein antibodies agreed well with the FTIR results and further demonstrated that the generation and maintenance of the gradient of cross-linked pectins, as well as the proportional distribution of arabinogalactan proteins in tube cell walls, are essential for pollen tube growth. These results strongly suggest that intracellular calcium release mediates the processes of pollen germination and pollen tube growth in P. wilsonii and its inhibition can lead to abnormal growth by disturbing the deposition of cell wall components in pollen tube tips.

To study the effect of chemical pretreatment on conifer resistance, 13-year-old Norway spruce (Picea abies (L.) Karst.) trees were treated with methyl jasmonate (MJ) or oxalic acid (OxA) on the outer bark and inoculated with the pathogenic blue-stain fungus Ceratocystis polonica (Siem.) C. Moreau 4 weeks later. Both chemicals significantly reduced symptoms of fungal infection, but MJ was more effective than OxA (51 versus 18% reduction in length of necrotic lesions in the phloem relative to untreated control trees). Anatomical examination of treated stem tissues showed that MJ induced extensive formation of traumatic resin ducts in the xylem and extra polyphenolic parenchyma (PP) cells in the secondary phloem between the cambium and the regular annual PP cell layer. No traumatic resin ducts were formed after treatment with OxA, and the coverage of extra PP cells in OxA-treated tissues was not significantly higher than in the controls. The anatomically based defense reactions induced by MJ were similar to the reactions observed after pathogen infection, mechanical wounding and bark beetle attack. Neither MJ nor OxA had apparent phytotoxic effects on Norway spruce at the concentrations used, with needle and stem tissues of all trees appearing normal without visible symptoms of toxicity. However, trees treated with MJ had 30% less radial sapwood growth than control trees. In conclusion, MJ treatment of Norway spruce appears to have practical potential as a tool for increasing plant resistance to fungal infection, but with a modest reduction in sapwood growth.

Several previous studies have observed that species and individuals with large seeds respond more positively to elevated CO (2) than those with small seeds. We explored the reasons for this pattern by examining the relationship between seed size and CO (2) response in Picea abies and P. rubens using growth analysis. The large seeded species (P. abies) responded more positively to elevated CO (2) than the small seeded species (P. rubens). At the intraspecific level, P. abies individuals from large seeds responded more positively to elevated CO (2) than individuals from small seeds, however, there was no significant intraspecific variation in CO (2) response in P. rubens. The greater CO (2) response of plants from large seeds was not simply the result of a larger starting capital compounded at the same rate as in plants from small seeds. Elevated CO (2) increased relative growth rate to a greater extent in individuals from large seeds. This effect appears to be related to differences in time of establishment, source to sink ratio and nutrient availability with seed size. These results are significant not only in understanding the potential effect of rising atmospheric CO (2) concentrations on plant populations, but also in understanding the factors affecting plant success at current atmospheric CO (2) levels due to the elevation of CO (2) within the litter layer that occurs at many germination sites.

Somatic embryogenesis (SE) is expected to play an important role in increasing productivity, sustainability, and uniformity of future US forests. For commercial use, SE technology must work with a variety of genetically diverse trees. Initiation in loblolly pine (Pinus taeda L.), the main commercial US forest species, is often recalcitrant for desirable genotypes. Liquid initiation medium with no or low gelling agent or placement of the explant on gelled medium followed later by a liquid medium overlay during the initiation process increased initiation for loblolly pine and Norway spruce (Picea abies). Loblolly pine liquid medium required reduction of NAA from 2 mg/l in gelled medium to 0.3 mg/l in liquid medium. Once the NAA concentration was adjusted, loblolly pine initiation occurred in liquid medium with fully immersed megagametophytes, explants supported at the liquid medium surface, or on gelled medium overlaid with liquid medium. Liquid overlays (0.25 ml) consisting of medium with NAA reduced to 0.3 mg/l, 9 mg/l ABA and no gelling agent applied to explants on 2 ml of gelled medium provided excellent initiation results. Greatest initiation percentages occurred when the liquid overlay was applied 14 days after placement of the megagametophyte on gelled medium. Initiation increases ranged from +8.5% with high-value cross-pollinated seed sources to +6.5 to +9.9% with open-pollinated and often recalcitrant seed sources. Liquid medium addition allows rapid replenishment of nutrients and adjustment or change of pH, hormones, or other parameters without disturbing the tissue.

Ozone is considered as the main factor in air pollution related to a decline of forest in North America and Europe. In the present study, the effect of changed litter quality, due to ozone stress to trees, on the microbial communities colonizing the subsequent litter was investigated. Litter bag technique using beech and spruce litter from ozone-stressed and control trees, was combined with 16S and 18S rRNA-based fingerprinting methods and cloning to characterize phylogenetic diversity. Litter bags were incubated for 2 and 8 weeks in a beech-spruce mixed forest. Differences between the structure of microbial communities colonizing control and ozone-exposed litter were evident by fingerprints of 16S and 18S rRNA RT-PCR products. RT-PCR products, from litter degraded for 8 weeks, were cloned to identify the bacterial and fungal groups. Clones similar to members of Actinobacteria dominated the bacterial libraries, whereas effects of changed litter quality were mainly observed for the Proteobacteria. Fungal libraries were dominated by clones similar to Ascomycota members. Reduced proportion of clones similar to Basidiomycota and Zygomycota in library from ozone-stressed spruce trees and Chytridiomycota from ozone-stressed beech trees was observed when compared to their control counterparts. As hypothesized, changed litter quality due to elevated O3 did influence the structure of litter-colonizing microbial communities. However, these differences were not as pronounced as those between the two plant species.

A cryopreservation method comprising sorbitol pre-growth treatment, DMSO cryoprotection and two-step controlled rate cooling has been optimized for differentiated embryogenic suspensor masses (ESM) of Picea sitchensis. The protocol was applied to clonal cultures from five different half-sibling families each represented by five different genotypes and their responses to cryopreservation assessed over 3 years. Nineteen of the 25 clonal lines tested survived LN and were capable of regrowth and producing stage 2-4 somatic embryos. Following the second subculture cycle of esm after they had been retrieved from cryogenic storage, post-cryopreservation regrowth was comparable with that of controls. A general linear model, multifactorial main effects plot revealed no significant differences between genotypes (p > 0.05) in response to cryopreservation, whereas significant differences between families (p < 0.05) were detected.

Epidemiological analysis of sequential growth data may be a tool in assessing ozone sensitivity of mature trees. Annual shoot growth of mature Fagus sylvatica in 83 Swiss permanent forest observation plots and of Picea abies in 61 plots was evaluated for 11 and 8 consecutive years, respectively, using branches harvested every 4 years. The data were assessed as annual deviation from average growth and related to fructification, ozone, meteorological parameters, and modelled soil water content using a mixed linear model. In beech, a significant association between ozone and shoot growth was observed which corresponded to a 7.4% growth reduction between 0 and 10 ppm h AOT40 (accumulated ozone over threshold 40). This is in the same order of magnitude as the response observed in experiments with seedlings. No interaction was found between ozone and drought parameters. In Norway spruce, shoot growth was neither associated with ozone nor with drought.

In white spruce (Picea glauca), an improvement of somatic embryo yield and quality can be achieved by applications of DL: -buthionine-[S,R]-sulfoximine (BSO), which inhibits the biosynthesis of reduced glutathione (GSH), thereby switching the total glutathione pool towards its oxidized form (GSSG). Applications of BSO almost tripled the embryogenic output of two cell lines by increasing the number of embryos produced by 100 mg(-1) tissue from 65 to 154 in the (E)WS1 line and from 59 to 130 in the (E)WS2 line. This increase in embryo number was ascribed to a higher production of morphologically normal embryos with four or more cotyledons (group A embryos), at the expense of group B embryos, characterized by fewer cotyledons. The quality of the embryos produced, estimated by their post-embryonic performance, was also different between treatments. In both cell lines applications of BSO in the maturation medium increased the conversion frequency, i.e. root and shoot emergence, of group A embryos while it enhanced root emergence in group B embryos. Compared to their control counterparts, BSO-treated embryos had normal shoot apical meristems as in their zygotic counterparts. Such meristems were characterized by large apical cells and vacuolated sub-apical cells. They also lacked intercellular spaces, which were present in the apical poles of control embryos where they contributed to cell-cell separation and meristem degradation. Furthermore, storage product accumulation was also improved in the presence of BSO, with protein bodies prevailing over starch. These data show that an oxidized glutathione environment is beneficial for spruce embryo production in vitro.

Tropospheric ozone (O3) levels are predicted to stay high, being a factor within "global change" with potential effects on the carbon sink strength of forest trees. Hence, new approaches to O3 risk assessment and their validation are required, although appropriate databases for adult trees are scant. Approaches based on external O3 exposure are presently being evaluated against the ones on O3 flux into leaves, as the cumulative uptake has the capacity for deriving O3 risk from cause-effect relationships. The effective dose, however, needs to account for the trees' O3 defence and tolerance in addition to O3 uptake. The current status of promoting the preferable mechanistic O3 flux concept is highlighted for major regions of Europe, addressing refinements and simplifications needed for routine use. At the pan-European scale, however, the flux-based concept is ready for use in O3 risk assessment and has the potential of meso-scale application at the forest ecosystem level.

Whole-tree O3 uptake was exemplified for Picea abies, Fagus sylvatica and Larix decidua in stands at high and low altitude and contrasting water availability through sap flow measurement in tree trunks, intrinsically accounting for drought and boundary layer effects on O3 flux. O3 uptake of evergreen spruce per unit foliage area was enhanced by 100% at high relative to low elevation, whereas deciduous beech and larch showed similar uptake regardless of altitude. The responsiveness of the canopy conductance to water vapor and, as a consequence, O3 uptake to soil moisture and air humidity did not differ between species. Unifying findings at the whole-tree level will promote cause-effect based O3 risk assessment and modeling.

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

In previous studies we have reported that applications of ascorbic acid (ACS) enhance the conversion frequency of white spruce somatic embryos by "rescuing" structurally disorganized meristems and inducing cell proliferation in the apical poles [C. Stasolla, E.C. Yeung, Ascorbic acid improves the conversion of white spruce somatic embryos, In Vitro Cell. Dev. Biol. Plant 35 (1999) 316-319]. In order to determine if the role played by this metabolite during embryo conversion is mediated by cellular peroxidases, the activity of guaiacol-, ferulic acid-, and ascorbic acid-dependent peroxidases were measured in the apical poles of germinating embryos with altered ASC levels. Changes in the endogenous ASC pool were achieved by treating the embryos with exogenously supplied ASC, L-galactono-gamma-lactone (GL) the last precursor of the de novo biosynthesis of ASC, and lycorine (L), an inhibitor of the last reaction leading to the synthesis of ASC. Our studies demonstrate the existence of a negative correlation between cellular ASC levels and activities of both guaiacol and ferulic acid peroxidases in root and shoot apices. A depletion of cellular ASC enhanced the rate of both guaiacol and ferulic acid oxidation at the apical poles of the embryos and resulted in meristem abortion. In contrast, the activity of guaiacol and ferulic acid peroxidases decreased below control levels if the endogenous ASC content of the embryos was experimentally increased. Fluctuations of total peroxidase activity following alterations in ASC pool were also confirmed by histochemical staining and in vitro studies. Overall our results suggest that a threshold of ASC level must be maintained in the apical poles of germinating embryos in order to inhibit peroxidase activities from cross-linking cell wall components and preventing post-embryonic growth.

The effects of exogenous abscisic acid (ABA) on the acclimation of Picea asperata to water deficit were investigated in two populations originating from wet and dry climate regions of China. Exogenous ABA was sprayed onto the leaves, and changes in plant growth and structure, gas exchange, water use efficiency (WUE), endogenous ABA content, and antioxidant enzyme levels were monitored. The results demonstrated that ABA application affected the two P. asperata populations in different ways during the water deficit. ABA application resulted in significantly lower CO(2) assimilation rates (A) under water deficit in plants from the wet climate population, whereas there were no significant changes in this parameter in the dry climate population. On the other hand, ABA application significantly decreased the dry shoot biomass, stomatal conductance (g(s)), transpiration rate (E), and malondialdehyde (MDA) content, and it significantly increased the leaf mass per area (LMA), root/shoot ratio (Rs), fine root/total root ratio (Ft), WUE, ABA content, and the superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) activities under water-deficit conditions in the dry climate population, whereas ABA application did not significantly affect these parameters in the wet climate population. The results clearly demonstrated that sensitivity to an exogenous ABA application is population-dependent in P. asperata. Direct evidence is presented that variation in physiological mechanisms rather than different rates of ABA absorption explain the population differentiation in the sensitivity to exogenous ABA, and that the physiological basis for the amplified response to water deficit caused by exogenous ABA, present mainly in the dry climate population, is related to internal ABA accumulation. These results provide evidence for adaptive differentiation between populations of P. asperata, and they support the expected relationship between environmental heterogeneity and the magnitude of plastic responses in plant populations.

Abscisic acid (ABA) plays an important role during somatic embryo development and maturation in coniferous species. The purpose of this research was to study ABA utilization by genotypes with different embryo maturation capabilities in interior spruce. Cell lines ISP11 and ISP48 were of high embryo maturation capability. By contrast, the tissue of line ISP16 contained numerous immature embryos, but only a few mature embryos developed. Exogenous ABA, i.e. S-ABA [(+)-cis, trans-ABA], racemic ABA, or ABA isomers were added into suspension cultures at a final concentration of 30 microM. In comparison to racemic ABA and ABA isomers, S-ABA reduced tissue proliferation the most. In all cell lines, about half of the racemic ABA was used within 2 weeks; the remaining ABA was (-)-cis, trans-ABA. The concentration of ABA showed little change thereafter. In the cultures supplied with ABA isomers, about half of (+/-)-cis, trans-ABA was utilized during 22 d. By contrast, (+/-)-trans, trans-ABA was hardly used, especially in line ISP16. S-ABA was almost completely metabolized by line ISP11. However, approximately 28% and 22% of the S-ABA remained in the culture of cell lines ISP16 and ISP48, respectively. Cell line ISP16 grew the fastest in culture. By 3 weeks, S-ABA consumption by ISP11 and ISP48 on the basis of tissue growth was, respectively, 2.2-fold and 3.4-fold greater than that of ISP16. A higher ratio of dihydrophaseic acid to phaseic acid existed with cell lines of higher embryo maturation capability, especially when the exogenously supplied ABA was chemically synthesized.

The broad range in plant responses to chronic O(3) exposure compels a search for integrative, underlying principles. One such approach is the unifying theory proposed by Reich (1987), which combines the O(3) response of contrasting physiognomic classes of plants on the basis of their intrinsic leaf diffusive conductance and, hence, capacity for O(3) uptake. Physiognomic classes differ in the proportional decline in photosynthesis and growth when compared on the basis of cumulative O(3) exposure per unit time, but converge when compared on the basis of O(3) uptake per unit time or cumulative O(3) uptake over the entire lifetime of the leaf. The theory is based on observations on a large number of contrasting plant species, relying primarily on studies of juvenile trees subjected to short-term O(3) exposure. To test the applicability of the unifying theory to mature trees, broadleaf deciduous European beech (Fagus sylvatica L.) and the evergreen conifer Norway spruce (Picea abies (L.) Karst.) in a mature mixed stand were exposed to either ambient air (control) or air with twice the ambient O(3) concentration delivered into the canopy by means of a free-air fumigation system. We accounted for differences in growing season length, leaf longevity and O(3)-related effects on leaf diffusive conductance in determining total O(3) uptake over the lifetime of the leaf. On this basis, Norway spruce needles required 5 years to take up as much O(3) as did beech leaves in one growing season. The core of the unifying theory on O(3) sensitivity was substantiated in relation to O(3) exposure and uptake. However, contrary to the unifying theory, which was formulated on the basis of results with juvenile trees, the O(3) response of mature trees in a natural stand was more complex. The increased complexity was attributed to additional environmental stressors, stress compensation at the whole-tree level, and differential O(3) sensitivities of leaves according to age class and position within the canopy. Contrary to the theory, photosynthesis was no less sensitive to O(3) in Norway spruce than that of beech, and was reduced in the twice-ambient O(3) regime in the first year of exposure.

Toxic effects of aluminium (Al) on Picea abies (L.) Karst. (Norway spruce) trees are well documented in laboratory-scale experiments, but field-based evidence is scarce. This paper presents results on fine root growth and chemistry from a field manipulation experiment in a P. abies stand that was 45 years old when the experiment started in 1996. Different amounts of dissolved aluminium were added as AlCl3 by means of periodic irrigation during the growing season in the period 1997-2002. Potentially toxic concentrations of Al in the soil solution were obtained. Fine roots were studied from direct cores (1996) and sequential root ingrowth cores (1999, 2001, 2002) in the mineral soil (0-40 cm). We tested two hypotheses: (1) elevated concentration of Al in the root zone leads to significant changes in root biomass, partitioning into fine, coarse, living or dead fractions, and distribution with depth; (2) elevated Al concentration leads to a noticeable uptake of Al and reduced uptake of Ca and Mg; this results in Ca and Mg depletion in roots. Hypothesis 1 was only marginally supported, as just a few significant treatment effects on biomass were found. Hypothesis 2 was supported in part; Al addition led to increased root concentrations of Al in 1999 and 2002 and reduced Mg/Al in 1999. Comparison of roots from subsequent root samplings showed a decrease in Al and S over time. The results illustrated that 7 years of elevated Al(tot) concentrations in the soil solution up to 200 microM are not likely to affect root growth. We also discuss possible improvements of the experimental approach.

An electrospray ionization mass spectrometric (ESI-MS) determination of glutathione (GSH), a sulfur-containing tripeptide (gamma-Glu-Cys-Gly) with regulation and detoxication functions in metabolisms of most living organisms, from nanomolar to micromolar levels is described. A hydrophilic interaction chromatography (HILIC) with an isocratic elution using a mobile phase containing acetonitrile and an aqueous 0.00005% solution of trifluoroacetic acid (60/40%, v/v) was applied for the separation of GSH. The peptide detection was achieved in the presence of L-ascorbic acid which significantly enhanced the signal intensity of the molecular ion GSH [M+H]+ (m/z 308). The calibration curve was linear (R2=0.9995) in the concentration range from 2 nM to 10 microM with a detection limit (LOD, S/N=3) of 0.5 nM. The excellent detection limit, and the excellent selectivity and high reproducibility of this method enabled determination of GSH in a single plant somatic embryo of a Norway spruce (Picea abies). The average amount of GSH in the single somatic embryos (n=18) was 9 pmol per embryo. Owing to our results, it can be supposed that the proposed HILIC/ESI-MS analysis might be used for GSH determination in microscopic cell structures and in single cell analyses.

Previously, we demonstrated that, in some cell lines, exogenous applications of ascorbic acid (ASC) enhance the conversion frequency of white spruce (Picea glauca (Moench) Voss) somatic embryos, by stimulating mitotic activity in the apical meristems. To examine this event in more detail, we investigated the effects of ASC on de novo, salvage and degradation pathways of pyrimidine metabolism by following the metabolic fate of (14)C-labeled orotic acid, thymidine, uridine and uracil in shoot and root poles of germinating embryos, after altering the cellular ASC content of the embryos. Alterations in endogenous ASC content did not affect the utilization of either orotic acid or uracil, but affected the metabolism of thymidine and uridine. Specifically, a lowering of endogenous ASC content by applications of lycorine (L), an inhibitor of the last enzyme of the ASC de novo biosynthetic pathway, resulted in a lower embryo conversion frequency, as well as a reduced percentage of thymidine and uridine incorporated into nucleotides and nucleic acids. The reduction in thymidine and uridine anabolism was mainly ascribed to the decreased activities of thymidine kinase (TRK) and uridine kinase (URK), the respective salvage enzymes of thymidine and uridine, measured in L-treated embryos. These effects were solely a result of a decrease in endogenous ASC content because applications of ascorbic acid plus lycorine (ASC + L) increased embryo conversion frequency, thymidine and uridine salvage activities, and TRK and URK activities to near control values. Inclusion of exogenous ASC in the germination medium did not affect the percentage of embryos able to convert to viable plantlets, although it increased thymidine and uridine utilization for nucleic acid synthesis in the shoot and root poles of the embryos. Taken together, these findings confirm that cellular ASC plays a key role in the reactivation of the apical meristems of germinating white spruce somatic embryos.

Black spruce (PICEA MARIANA), white spruce (PICEA GLAUCA), and jack pine (PINUS BANKSIANA) seedlings were inoculated with HEBELOMA CRUSTULINIFORME or LACCARIA BICOLOR and subjected to NaCl and Na (2)SO (4) treatments. The effects of ectomycorrhizas on salt uptake, growth, gas exchange, and needle necrosis varied depending on the tree and fungal species. In jack pine seedlings, ectomycorrhizal (ECM) fungi reduced shoot and root dry weights and in the ECM white spruce, there was a small increase in dry weights. Sodium chloride treatment reduced net photosynthesis and transpiration rates in the three studied tree species. However, NaCl-treated black spruce and jack pine colonized by H. CRUSTULINIFORME maintained relatively high photosynthetic and transpiration rates and needle necrosis of NaCl-treated black spruce seedlings was reduced by the ECM fungi. Higher concentrations of Na (+) were found in shoots compared with roots of the three examined conifer species. ECM fungi reduced the concentrations of Na (+) mainly in the shoots and this reduction was greater in plants treated with NaCl compared with Na (2)SO (4). Shoots contained generally higher concentrations of Cl (-) compared with roots. In the NaCl-treated black spruce and white spruce, both ECM species significantly reduced Cl (-) concentrations. Our results point to overall greater phytotoxicity of NaCl compared with Na (2)SO (4) and support our earlier findings which demonstrated beneficial effects of ECM fungi for woody plants exposed to NaCl stress.

We tested the effects of ectomycorrhizal (ECM) inoculation on greenhouse-grown white spruce (Picea glauca (Moench) Voss) and jack pine (Pinus banksiana L.) seedlings to be used for revegetation of salt-affected tailing sands resulting from the exploitation of oil sand in northeastern Alberta, Canada. White spruce and jack pine seedlings were inoculated with three ECM fungi selected for their in vitro tolerance to excess Na+ and Cl-: Hebeloma crustuliniforme (Bull) Quel. UAMH 5247, Laccaria bicolor Maire (Orton) UAMH 8232 and a Suillus tomentosus (Kauff.) Sing., Snell and Dick isolate from a salt-affected site. The physiological responses of the seedlings to a gradient of NaCl concentration (0, 50, 100 and 200 mM) were assessed over four weeks by: (1) Na+ accumulation and allocation; (2) chlorophyll a fluorescence; (3) growth, (4) water content; and (5) organic osmolyte accumulation. Jack pine seedlings were more sensitive than white spruce seedlings to increasing Na+ and Cl- concentrations. Both species showed decreasing biomass accumulation, and increasing concentrations of organic osmotica and Na with increasing NaCl concentration. White spruce seedlings inoculated with the S. tomentosus isolate had the best growth response at all NaCl concentrations tested. Although jack pine seedlings inoculated with the L. bicolor or S. tomentosus isolate exhibited the highest growth in the 50 and 100 mM NaCl treatments, both fungi increased the photochemical stress and dehydration of their hosts in the 200 mM NaCl treatment. At the latter concentration, jack pine seedlings inoculated with H. crustuliniforme showed the greatest tolerance to salt stress. Although the different fungi altered the physiological response of the host in different ways, inoculation with salt-stress-tolerant ECM fungi increased growth and reduced the negative effects of excess NaCl. Use of controlled mycorrhization may increase survival of coniferous seedlings used for revegetation of salt-affected sites.

Evanescent wave excitation was used to visualize individual, FM4-64-labeled secretory vesicles in an optical slice proximal to the plasma membrane of Picea meyeri pollen tubes. A standard upright microscope was modified to accommodate the optics used to direct a laser beam at a variable angle. Under evanescent wave microscopy or total internal reflection fluorescence microscopy, fluorophores localized near the surface were excited with evanescent waves, which decay exponentially with distance from the interface. Evanescent waves with penetration depths of 60 to 400 nm were generated by varying the angle of incidence of the laser beam. Kinetic analysis of vesicle trafficking was made through an approximately 300-nm optical section beneath the plasma membrane using time-lapse evanescent wave imaging of individual fluorescently labeled vesicles. Two-dimensional trajectories of individual vesicles were obtained from the resulting time-resolved image stacks and were used to characterize the vesicles in terms of their average fluorescence and mobility, expressed here as the two-dimensional diffusion coefficient D2. The velocity and direction of vesicle motions, frame-to-frame displacement, and vesicle trajectories were also calculated. Analysis of individual vesicles revealed for the first time, to our knowledge, that two types of motion are present, and that vesicles in living pollen tubes exhibit complicated behaviors and oscillations that differ from the simple Brownian motion reported in previous investigations. Furthermore, disruption of the actin cytoskeleton had a much more pronounced effect on vesicle mobility than did disruption of the microtubules, suggesting that actin cytoskeleton plays a primary role in vesicle mobility.

When conifers such as Picea abies Karst. (Norway spruce) are attacked by insects or pathogens, they often respond by producing increased quantities of terpenoid oleoresin. This response can be mimicked in young P. abies seedlings by treatment with methyl jasmonate (MJ). In this study, we determined the effects of MJ on terpenoids and other chemical defenses of mature P. abies, and investigated whether this treatment protected trees against attack by the blue-stain fungus Ceratocystis polonica (Siem.) C. Moreau, the most important fungal associate of the spruce bark beetle Ips typographus L. Methyl jasmonate treatment induced the formation of traumatic resin ducts in the developing xylem, enhanced resin flow and stimulated increased accumulation of monoterpenes, sesquiterpenes and diterpene resin acids. However, only minor changes were detected in terpene composition in response to MJ treatment and no changes in soluble phenolic concentration were measured. There was much variability in the timing and degree of response to MJ among clones. The observed chemical and anatomical changes in response to MJ treatment were correlated with increased resistance to C. polonica, suggesting that terpenoid oleoresin may function in defense against this pathogen.

To investigate roles of the actin cytoskeleton in growth of the pollen tube of Picea meyeri, we used the actin polymerization inhibitor latrunculin B (LATB) under quantitatively controlled conditions. At low concentrations, LATB inhibited polymerization of the actin cytoskeleton in the growing pollen tube, which rapidly inhibited tip growth. The proteomic approach was used to analyse protein expression-profile changes during pollen germination and subsequent pollen-tube development with disturbed organization of the actin cytoskeleton. Two-dimensional electrophoresis and staining with Coomassie Brilliant Blue revealed nearly 600 protein spots. A total of 84 of these were differentially displayed at different hours with varying doses of LATB, and 53 upregulated or downregulated proteins were identified by mass spectrometry. These proteins were grouped into distinct functional categories including signalling, actin cytoskeleton organization, cell expansion and carbohydrate metabolism. Moreover, actin disruption affected the morphology of Golgi stacks, mitochondria and amyloplasts, along with a differential expression of proteins involved in their functions. These findings provide new insights into the multifaceted mechanism of actin cytoskeleton functions and its interaction with signalling, cell-expansion machinery and energy-providing pathways.

We assessed the effects of brefeldin A (BFA) on pollen tube development in Picea meyeri using fluorescent marker FM4-64 as a membrane-inserted endocytic/recycling marker, together with ultrastructural studies and Fourier transform infrared analysis of cell walls. BFA inhibited pollen germination and pollen tube growth, causing morphological changes in a dose-dependent manner, and pollen tube tip growth recovered after transferring into BFA-free medium. FM4-64 labeling showed typical bright apical staining in normally growing P. meyeri pollen tubes; this apical staining pattern differed from the V-formation pattern found in angiosperm pollen tubes. Confocal microscopy revealed that exocytosis was greatly inhibited in the presence of BFA. In contrast, the overall uptake of FM4-64 dye was about 2-fold that in the control after BFA (5 microg mL(-1)) treatment, revealing that BFA stimulated endocytosis in a manner opposite to the induced changes in exocytosis. Transmission electron microscopic observation showed that the number of secretory vesicles at the apical zone dramatically decreased, together with the disappearance of paramural bodies, while the number of vacuoles and other larger organelles increased. An acid phosphatase assay confirmed that the addition of BFA significantly inhibited secretory pathways. Importantly, Fourier transform infrared microspectroscopy documented significant changes in the cell wall composition of pollen tubes growing in the presence of BFA. These results suggest that enhanced endocytosis, together with inhibited secretion, is responsible for the retarded growth of pollen tubes induced by BFA.

This study attempted to detect the impact of ozone on adult trees of Norway spruce ( Picea abies [L.] Karst.) and European beech ( Fagus sylvatica L.) in an experimental mixed stand in Southern Bavaria, Germany. The aim was to examine whether there is a decrease in growth when trees are exposed to higher than atmospheric concentrations of ozone. This exposure was put into effect using a free-air fumigation system at tree crown level. Growth analysis was carried out on a group of 47 spruce and 36 beech trees, where radial stem increment at breast height - a sensitive index for stress - was measured. The ozone monitoring system allowed values to be obtained for the accumulated ozone exposure (SUM00) of each individual tree, so that their radial increment over three years could be correlated with the corresponding ozone exposure for the same time period. Correlation and regression analysis were then carried out to test the influence of ozone on diameter increment. In both spruce and beech, the initial stem diameter was the most influential factor on radial increment in the following year. A linear model was applied, including the diameter of the preceding year and the ozone exposure of the current year as predicting factors. For spruce trees, a significant negative influence of ozone exposure was found. In contrast, no significant ozone effect on diameter increment of beech was detected. The effect of ozone stress on a large spruce tree can lead to a decrease in potential radial increment of 22 %. The results are discussed in relation to other stress factors such as drought and lack of light.