Effects of ozone on starch accumulation in Norway spruce (Picea abies)

Summary Fumigation with 100 g/m³ and 200 g/m³ ozone in closed-top fumigation chambers induced starch accumulation in chloroplasts of Norway spruce. This accumulation was probably due to a partial inhibition of the starch translocation at night. The intensity of the effect was dependent on the season and the age of the needles. The accumulation was reversed in winter. It is therefore unlikely that such an effect has much significance for plant health.     

Enhancement of somatic embryogenesis in Norway spruce (Picea abies L.)

Summary Embryogenic callus developed in 55% of the mature embryo explants of Norway spruce (Picea abies L.) growing on a LP medium minus the amino acids and sugars (except sucrose). This is the highest reported yield of embryogenic callus from mature embryos of P. abies that has ever been reported. Callus induction from either the middle or the end of the hypocotyl of the embryos began after 2–3 weeks. Three types of calli were recovered: (a) globular, (b) light green-compact, (c) white mucilaginous. Only the white mucilaginous calli were embryogenic. The globular and light green-compact calli never become embryogenic, even after several subcultures. The development of somatic embryos was accomplished on half-strength macro-elements of NSIII medium containing 1 M -naphthaleneacetic acid, 1 M abscisic acid, and 3% sucrose. The addition of 10^–7 M buthionine sulfoximine to the medium increased the development of somatic embryos by three fold. These results suggest that there is a great potential for increasing the frequency and development of somatic embryos in P. abies. Careful selection of the genotype and modification of the culture medium is required.     

Elicitor-mediated induction of enzymes of lignin biosynthesis and formation of lignin-like material in a cell suspension culture of spruce (Picea abies)

Incubations of photomixotrophic suspension culture cells of spruce (Picea abies) (L.) (Karst) with an autoclaved cell wall preparation of Rhizosphaera kalkhoffii as elicitor led to a rapid increase of the activity of a number of enzymes involved in lignin biosynthesis. l-phenylalanine ammonia-lyase (EC 4.3.1.5) was induced about 10-fold, feruloyl-Coenzyme A reductase (ED 1.2.1.44) 4-fold, cinnamyl alcohol dehydrogenase (NADP⁺) (EC 1.1.1.195) 2-fold and peroxidase (EC 1.11.1.7) about 1.5-fold. The induction of the enzymes, with the exception of the peroxidase, was transient, showing maximal activity within 3 days after elicitation. Extracellular peroxidase activity, determined in the culture medium, rapidly decreased on initiation of elicitation.Concomitant with the increase of activity of the enzymes of lignin synthesis was a rapid clouding of the culture medium. Phloroglucinol-HCl staining revealed the presence of lignin-like material in the medium and also in the cells. The IR-spectrum of this material was identical with the IR-spectrum of authentic spruce lignin.Abbreviations PAL l-phenylalanine ammonia-lyase - FCR feruloyl-Coenzyme A reductase - CAD cinnamyl alcohol dehydrogenase - POD peroxidase     

l-Phenylalanine ammonia-lyase in suspension culture cells of spruce (Picea abies)

The activity of l-phenylalanine ammonia-lyase (PAL) (EC 4.3.1.5) was determined in seedlings, callus cells, cell suspension cultures and in young needles of spruce (Picea abies) (L.) (Karst). PAL activity increased up to 10 fold in response to transferring suspension cultured cells into new cultivation medium. PAL was also induced about 10 fold when callus cells were transferrd into liquid medium. The increase was transient and it required the presence of a carbohydrate.In cell suspension cultures, grown in the dark (white cells), but not in light-grown cultures (green cells), PAL activity was induced up to 30 fold by UV-light.With a cell wall preparation of Rhizosphaera kalkhoffii, a forest pathogenic fungus, used as elicitor, the activity of PAL could be induced more than 10 fold. The degree of induction depended on the elicitor concentration. Induction was prevented by cycloheximide but not by actinomycin D.     

Diterpene resin acid biosynthesis in loblolly pine (Pinus taeda): functional characterization of abietadiene/levopimaradiene synthase (PtTPS-LAS) cDNA and subcellular targeting of PtTPS-LAS and abietadienol/abietadienal oxidase (PtAO, CYP720B1)

Diterpene resin acids are prominent defense compounds against insect pests and pathogens in conifers. Biochemical and molecular analyses in grand fir (Abies grandis), Norway spruce (Picea abies), and loblolly pine (Pinus taeda) have identified two classes of genes and enzymes that generate much of the structural diversity of terpenoid defense compounds: The terpenoid synthases (TPS) and cytochrome P450 monooxgenases (P450). Using a single substrate, geranylgeranyl diphosphate, families of single-product and multi-product diterpene synthases generate an array of cyclic diterpene olefins. These diterpenes are converted to diterpene resin acids by activity of one or more P450 enzymes. A few conifer diterpene synthases have previously been cloned and characterized in grand fir and in Norway spruce. We have also previously shown that the loblolly pine P450 abietadienol/abietadienal oxidase (PtAO) catalyzes multiple oxidations of several diterpene alcohols and aldehydes. Conifer diterpene synthases are thought to function in plastids while P450s can also be localized to plastids or to the endoplasmic reticulum (ER). Here, we show that a loblolly pine cDNA (PtTPS-LAS) encodes a typical multi-product conifer diterpene synthase that forms levopimaradiene, abietadiene, palustradiene, and neoabietadiene similar to the grand fir abietadiene synthase and Norway spruce levopimaradiene/abietadiene synthase. Subcellular targeting of PtTPS-LAS and PtAO to plastids and ER, respectively, was shown with green fluorescent fusion protein expression in tobacco cells. These data suggest that enzymes for conifer diterpene resin acid biosynthesis are localized to at least two different subcellular compartments, plastids and ER, requiring efficient transport of intermediates and secretion of diterpene resin acids into the extracelluar space.     

Biochemical symptoms of stress in the mycorrhizal roots of Norway spruce (Picea abies)

Summary The mycorrhizal activity of spruce in a mixed-wood forest was monitored over 1 year by measuring biochemical characters in fine roots of six canopy trees and of a regrowth stand. The concentration of adenosine 5-triphosphate (ATP), a measure of living biomass, showed two peaks per year, one at bud break and one after main shoot growth. The concentration of storage polysaccharides in mycorrhizae showed the same cycles even more pronouncedly. It is proposed that these changes reflect growth and senescence of mycorrhizae and that the timing of the cycles is controlled by translocation of assimilates from the shoot. Differences between mycorrhizae collected from canopy trees and the regrowth stand were small and not significant. Characters known to be related to fungal activity of the mycorrhizal symbiosis (concentration of trehalose, glucose uptake, respiration) also varied little among the six canopy trees. Large differences among fine-root samples from different canopy trees, however, were detected in the concentrations of ATP and storage polysaccharides, measures which seemed to be physiologically integrated within trees. If low concentrations in roots precede losses of foliage from trees, these two symptoms could be used as early indicators of growth decline in individual spruce trees.     

Triacylglycerols in the seeds of northern Scots pine,Pinus sylvestris L., and Norway spruce,Picea abies (L.) Karst.

The geographical variation in the composition of triacylglycerols in seeds of Scots pine (Pinus sylvestris L.) and Norway spruce [Picea abies (L.) Karst.], grown in Finland, was analyzed. The total lipid content of pine seeds was slightly higher in the northernmost provenance (68 °50N), whereas the lipid content of spruce seeds was not affected systematically by the geographical origin of the seeds. The species studied differed in the proportions of fatty acids in their triacylglycerols, though the three most abundant components were the same, i.e. oleic acid (181 n9), linoleic acid (182n-6) and 5,9,12-octadecatrienoic acid (183 5c9c12c). These fatty acids corresponded to more than 80 mol% of the total fatty acids. According to mass spectrometric analyses, the triacylglycerols of both spruce and pine seeds consisted of the same molecular species with 52–56 acyl carbons, but in different proportions. Molecules with 54 acyl carbons represented approximately 75% of the pine and 85% of the spruce triacylglycerols, with the most abundant molecular species being 545, 546 and 547. Some minor differences in the fatty acid composition of triacylglycerols of pine seeds from different seed collecting areas were found: the proportion of linoleic acid slightly increased whereas that of 5,11,14-eicosatrienoic acid decreased towards the northern origins. Similar differences were not found in the proportions of fatty acids in spruce seed lots. Furthermore, the proportions of triacylglycerols in both pine and spruce seeds from northern and southern collecting areas were not significantly different. The higher content of total lipids in spruce seeds compared with pine seeds may be due to the structure of the seed coat, and the lipophilic layers inside it, acting as a barrier to imbibition.     

Multivariate interpretation of the foliar chemical composition of Norway spruce (Picea abies)

Twenty-four chemical elements were analysed by INAA, ICP-AES and CN in needles of Norway spruce (Picea abies (L.) Karst.). Branches were sampled from 54 trees on eight sites in Switzerland and South Germany. From each tree, twigs were sorted into the most recent four or five age classes and their needles analysed separately. All measured concentrations could be considered as log-normally distributed and statistical analyses were, therefore, performed on logarithms. Variance components were estimated by maximum likelihood and compared between elements. Non-essential elements varied more than essential nutrients (Mn was an exception). The sites and the age of the needles were the most important sources of variance. The interaction between site and age, the individual tree, the sampled branch and the residual variance were usually much smaller sources of variance. The effects of the most significant factors – site and age – were further described by principal components and cluster analyses. Mineral elements either increased or decreased with the age of the needles according to their mobility in the phloem. Two different components were identified in the effect of the sites: a geochemical component linked to soil pH and a climate component linked to altitude, temperature and precipitation. Multivariate statistics are discussed as a tool for the interpretation of complex interaction patterns between element concentrations in plants.     

Molecular characterization of a phosphoenolpyruvate carboxylase in the gymnosperm Picea abies (Norway spruce)

Phosphoenolpyruvate carboxylase (PEPC) genes and cDNA sequences have so far been isolated from a broad range of angiosperm but not from gymnosperm species. We constructed a cDNA library from seedlings of Norway spruce (Picea abies) and identified cDNAs coding for PEPC. A full-length PEPC cDNA was sequenced. It consists of 3522 nucleotides and has an open reading frame (ORF) that encodes a polypeptide (963 amino acids) with a molecular mass of 109 551. The deduced amino acid sequence revealed a higher similarity to the C3-form PEPC of angiosperm species (86–88%) than to the CAM and C4 forms (76–84%). The putative motif (Lys/Arg-X-X-Ser) for serine kinase, which is conserved in all angiosperm PEPCs analysed so far, is also present in this gymnosperm sequence. Southern blot analysis of spruce genomic DNA under low-stringency conditions using the PEPC cDNA as a hybridization probe showed a complex hybridization pattern, indicating the presence of additional PEPC-related sequences in the genome of the spruce. In contrast, the probe hybridized to only a few bands under high-stringency conditions. Whereas this PEPC gene is highly expressed in roots of seedlings, a low-level expression can be detected in cotyledons and adult needles. A molecular phyiogeny of plant PEPC including the spruce PEPC sequence revealed that the spruce PEPC sequence is clustered with monocot and dicot C3-form PEPCs including the only dicot C4 form characterized so far.     

The spermatogenous body cell of the coniferPicea abies (Norway spruce) contains actin microfilaments

Summary In conifer pollen, the generative cell divides into a sterile stalk cell and a body cell, which subsequently divides to produce two sperm. InPicea abies (Norway spruce, Pinaceae) this spermatogenous body cell contains actin microfilaments. Microfilament bundles follow the spherical contour of the body cell within the cell cortex, and also traverse the cytoplasm and enmesh amyloplasts and other organelles. In addition, microfilaments are associated with the surface of the body cell nucleus. The sterile stalk cell also contains microfilament bundles in the cytoplasm, around organelles, and along the nuclear surface. Within the pollen grain, microfilament bundles traverse the vegetative-cell cytoplasm and are enriched in a webbed cage which surrounds the body cell. Microfilaments were identified with rhodamine-phalloidin and with indirect immunofluo-rescence using a monoclonal antibody to actin. The majority of evidence in literature suggests that the spermatogenous generative cell in angiosperms does not contain actin microfilaments, so the presence of microfilaments within the spermatogenous body cell inP. abies appears to be a fundamental difference in sexual reproduction between conifers and angiosperms.     

Microtubules and microfilaments are both responsible for pollen tube elongation in the coniferPicea abies (Norway spruce)

Summary InPicea abies (Norway spruce), microtubules and actin microfllaments both form a dense matrix throughout the tube mainly parallel to the direction of elongation. In these conifer pollen tubes the organization of this matrix is different from that in angiosperms. This study tests our hypothesis that differences in cytoskeletal organization are responsible for differences in tube growth and physiology. Pollen grains were germinated in media containing cytoskeletal disrupters and analyzed for germination, tube length, tube branching, and tip swelling. Disruption of microtubules significantly inhibits tube elongation and induces tube branching and tip swelling. Tip swelling is probably caused by disruption of the microtubules in the tip that are perpendicular to the direction of elongation. Confocal microscopy indicates that colchicine and propyzamide cause fragmentation of microtubules throughout the tube. Oryzalin and amiprophosmethyl cause a complete loss of microtubules from the tip back toward the tube midpoint but leave microtubules intact from the midpoint back to the grain. Disruption of microfilaments by cytochalasins B and D and inhibition of myosin by N-ethylmaleimide or 2,3-butanedione monoxime stops tube growth and inhibits germination. Microfilament disruption induces short branches in tubes, probably originating from defective microfilament organization behind the tip. In addition, confocal microscopy coupled with microinjection of fluorescein-labeled phalloidin into actively growing pollen tubes indicates that microfllament bundles extend into the plastid-free zone at the tip but are specifically excluded from the growing tip. We conclude that microtubules and microfilaments coordinate to drive tip extension in conifer pollen tubes in a model that differs from angiosperms.     

Respiration and photosynthesis in cones of Norway spruce (Picea abies (L.) Karst.)

Summary Dark respiration and photosynthetic carbon dioxide refixation in purple and green Picea abies cones were investigated from budbreak to cone maturity. The rate of dark respiration per unit dry weight and CO₂ refixation capacity decreased during cone maturation. At the beginning of the growing season, photosynthetic CO₂ refixation could reduce the amount of CO₂ released by respiration in green and purple cones by 50% and 40%, respectively. The seasonal performance of the components of the cone carbon balance was calculated using information on the seasonal course of respiration, refixation capacity and the light response curves of cone photosynthesis, as well as the actual light and temperature regime in the field. The daily gain of CO₂ refixation reached 28%–34% of respiration in green and 22%–26% in purple cones during the first month of their growth, but decreased later in the season. Over the entire growth period refixation reduced carbon costs of cone production in both cone colour polymorphs by 16%–17%.     

Nitrogen limitation in mycorrhizal Norway spruce (Picea abies) seedlings induced mycelial foraging for ammonium: implications for Ca and Mg uptake

Although many studies support the importance of the external mycelium for nutrient acquisition of ectomycorrhizal plants, direct evidence for a significant contribution to host nitrogen nutrition is still scarce. We grew nonmycorrhizal seedlings and seedlings mycorrhizal with Paxillus involutus (Batsch) Fr. in a sand culture system with two compartments separated by a 45-m Nylon mesh. Hyphae, but not roots, can penetrate this net. Nutrient solutions were designed to limit seedling growth by nitrogen. Hyphal density in the hyphal compartment, host N status and shoot growth of mycorrhizal seedlings significantly increased in response to NH₄ ⁺ addition to the hyphal compartment. Labeling the compartment only accessible to hyphae with ¹⁵NH₄ ⁺ showed that the increase in N uptake in the mycorrhizal seedlings was a result of hyphal N acquisition from the hyphal compartment. These results indicate that hyphae of P. involutus may actively forage into N-rich patches and improve host N status and growth. In the mycorrhizal seedlings, which received additional NH₄ ⁺ via their external mycelium, the increase in NH₄ ⁺ supply less negatively affected Ca and Mg uptake than in nonmycorrhizal seedlings, where the additional NH₄ ⁺ was directly supplied to the roots. This was most likely due to the close link of NH₄ ⁺ uptake and H⁺ extrusion, which, in the nonmycorrhizal seedlings, lead to a strong acidification in the root compartment, and subsequently reduced Ca and Mg uptake, whereas in the mycorrhizal seedlings the site of intensive NH₄ ⁺ uptake and acidification was in the hyphal and not in the root compartment. Our data support the idea that the ectomycorrhizal mycelium connected to an N-deficient host may actively forage for N. The mycelium may also be important as a biological buffer system ameliorating negative influence of high NH₄ ⁺ supply on cation uptake.     

Cloning, characterization and localization of three novel class III peroxidases in lignifying xylem of Norway spruce (Picea abies)

Plant class III peroxidases (POXs) take part in the formation of lignin and maturation of plant cell walls. However, only a few examples of such peroxidases from gymnosperm tree species with highly lignified xylem tracheids have been implicated so far. We report here cDNA cloning of three xylem-expressed class III peroxidase encoding genes from Norway spruce (Picea abies). The translated proteins, PX1, PX2 and PX3, contain the conserved amino acids required for heme-binding and peroxidase catalysis. They all begin with putative secretion signal propeptide sequences but diverge substantially at phylogenetic level, grouping to two subclusters when aligned with other class III plant peroxidases. In situ hybridization analysis on expression of the three POXs in Norway spruce seedlings showed that mRNA coding for PX1 and PX2 accumulated in the cytoplasm of young, developing tracheids within the current growth ring where lignification is occurring. Function of the putative N-terminal secretion signal peptides for PX1, PX2 and PX3 was confirmed by constructing chimeric fusions with EGFP (enhanced green fluorescent protein) and expressing them in tobacco protoplasts. Full-length coding region of px1 was also heterologously expressed in Catharanthus roseus hairy root cultures. Thus, at least the spruce PX1 peroxidase is processed via the endoplasmic reticulum (ER) most likely for secretion to the cell wall. Thereby, PX1 displays correct spatiotemporal localization for participation in the maturation of the spruce tracheid secondary cell wall.     

Tissue-specific expression of Pa18, a putative lipid transfer protein gene,during embryo development in Norway spruce (Picea abies)

A full-length Picea abies cDNA clone Pa18, encoding a protein with the characteristics of plant lipid transfer proteins, has been isolated and characterized. The size of the deduced 173 amino acid (aa) long protein is around 18 kDa. The first 100–120 aa show similarity to angiosperm lipid transfer proteins in amino acid sequence as well as in predicted secondary structure. The Pa18 gene is constitutively expressed in embryogenic cultures of Picea abies representing different stages of development as well as in non-embryogenic callus and seedlings. The Pa18 gene product has an antimicrobial activity. In situ hybridization showed that the Pa18 gene is equally expressed in all embryonic cells of proliferating embryogenic cultures but during embryo maturation the expression of the gene in maturing and mature somatic as well as in mature zygotic embryos is stronger in the outer cell layer than in other tissues. Southern blot analysis at different stringencies was consistent with a single gene with one or two copies rather than a gene family. Twenty independent transgenic sublines over- and under-expressing the Pa18 gene under the Zea mays ubiquitin promoter were established. There was a high yield of mature somatic embryos with a smooth surface only in untransformed, control cultures. Irrespective of the expression level of Pa18, the somatic embryos started to mature when given a maturation treatment. However, in the transgenic sublines, the outer cells in the maturing embryos frequently became elongated and vacuolated instead of remaining small and uniform. One explanation for this was that the expression of Pa18 was not restricted to the outer cell layer in transformed sublines. Angiosperms and gymnosperms separated about 300 million years ago and the embryo genesis is different in the two groups. The outer cell layer (protoderm), the first tissue to differentiate, is less clearly delineated in gymnosperms. For normal embryo development in angiosperms, expression of the LTP gene must be restricted to the protodermal cells. In this work we show that the expression of the Pa18 gene must be restricted to the putative protodermal cells of the gymnosperm.     

Stomatal SO2 uptake and sulfate accumulation in needles of Norway spruce stands (Picea abies) in Central Europe

Monthly uptake rates and the annual deposition of gaseous SO₂ via the stomata of six Norway spruce canopies (Picea abies (L.) Karst.) in Germany (Königstein im Taunus, Witzenhausen, Grebenau, Frankenberg, Spessart, Fürth im Odenwald) were calculated (i) from statistical response functions of stomatal aperture depending on meteorological data, and (ii) from the synchronously measured SO₂ immission at these stands. The stomatal response functions had been derived on the basis of thorough stomatal water conductance measurements in the field. Calculations of the SO₂ conductance of spruce twigs and SO₂ uptake rates via stomata need continuously measured complete data sets of the (i) light intensity, (ii) air temperature, (iii) air humidity and (iv) SO₂ concentration in spruce forests from all the year. These data were recorded half hourly in different German spruce forests. The apparent needle water vapour pressure difference and transpiration rates were calculated from meteorological data. Additional use of canopy through flow data in dry years allowed the estimation of the mean stomatal conductance for H₂O and SO₂ of whole spruce canopies. The annual SO₂ uptake of a mean unit needle surface in spruce forests was 32% of the SO₂ uptake rate of exposed needles at the top of spruce crowns. There is significant SO₂ uptake all the year. The mean SO₂ dose at all sites and years received through the stomata was (0.25±0.07) mol SO₂ m^-2 (total needle surface) (nPa Pa^-1)^-1 (annual mean of SO₂ immission; 1 nPa (SO₂) Pa^-1 (air) = 1 ppb) day^-1 (vegetation period per year). Comparison of calculated SO₂ uptake rates into needles with measured SO₄ ^2- accumulation rates in needles from the mentioned sites and additionally from Würzburg, Schneeberg (Fichtelgebirge) and from three sites in the eastern Erzgebirge (Höckendorf, Kahleberg, Oberbärenburg) revealed that oxidative SO₂ detoxification (SO₄ ^2- formation) dominates only at sites with high SO₂ immission and short vegetation periods. Under these conditions 70 to 90% of the annual stomatal SO₂ uptake is detoxified via SO₄ ^2- accumulation in needles. Cations are needed for neutralization of accumulating SO₄ ^2- which are inavailable to support growth. Thus, SO₂ induces a dominant and competitive additional nutrient cation demand, cation deficiency symptoms and enhanced needle loss (spruce decline symptoms) mainly at sites, where the ratio R=(SO₂ immission): (length of the vegetation period) is higher than R=0.07 nPa Pa^-1 day^-1. Correlation analysis of the relative needle loss versus the SO₂-dependent SO₄ ^2- formation rate revealed a significant increase of needle loss at the 98% level (Student). At sites with small SO₂ immission and long vegetation periods (R<0.07 nPa Pa^-1 day^-1) reductive SO₂ detoxification via growth (and/or phloem export of SO₄ ^2-) is not kinetically overburdened. Under these conditions only 30% of the annual SO₂ uptake is detoxified via SO₄ ^2- formation and spruce decline is small or absent. On the basis of the critical value R0.07 nPa Pa^-1 day^-1 recommended SO₂ immission limits can be deduced on a mere ecophysiological basis. These deduced values are close to the proposed SO₂ immission limits of the IUFRO, WHO and the UNECE.