Karyotype of Norway spruce by multicolor FISH

The chromosomes (2n = 2x = 24) of Norway spruce are very large since their size reflects the huge amount of genomic DNA (2C = 30 × 10⁹ bp). However, the identification of homologous pairs is hampered by their high degree of similarity at the morphological level. Data so far presented in the literature were not sufficient to solve all the ambiguities in chromosome identification. Several genomic Norway spruce DNA clones containing highly repetitive sequences have been identified and characterised in our laboratory. Three of them were selected for fluorescent in situ hybridization (FISH) experiments because of their strong signals and suitability for chromosome identification: PATR140 hybridized at the centromeric site of three chromosome pairs; PAF1 hybridized in six subtelomeric and two centromeric sites; 1PABCD6 co-localized with the subtelomeric sites identified by PAF1. The statistical analysis of microscopic measurements of chromosomes in combination with the FISH signals of these probes allowed the unambigous construction of Norway spruce karyotype. We also compared the karyotype of Norway spruce with that of other spruce species to infer the number and kind of rearrangements that have occurred during the evolution of these species.Communicated by D.B. Neale     

Callus cultures and bark from Norway spruce clones show similar cellular features and relative resistance to fungal pathogens

In field experiments, clones of Norway spruce [Picea abies (L.) Karst.] showed different degrees of resistance against pathogenic fungi inoculated into the bark that correlate with differences in polyphenolic parenchyma (PP) cells of the bark. Cells of spruce callus cultures, particularly towards the callus surface, resemble PP cells and this study looks at changes in callus cells during infection and the relative resistance of cultures from clones of low (weak) or high (strong) resistance to fungal infection. Callus cultures, initiated from trees with different resistance, were co-inoculated with Ceratocystis polonica (Siem.) C. Moreau and Heterobasidion annosum (Fr.) Bref. Callus cells from strong clones resemble PP cells of bark tissue from strong clones, having more polyphenolic bodies, while callus cells from weak clones are more similar to PP cells from those clones, which have less extensive phenolic bodies. Callus cultures from trees with weak resistance were more quickly overgrown by both species of pathogenic fungi than cultures from trees with strong resistance. Callus cells of infected cultures showed changes similar to activated PP cells of bark, including enhanced accumulation of polyphenolics. Phenolic bodies were more numerous and more extensive (larger and denser) in callus cells of strong versus weak clones under all conditions. Thus, callus cells may perform similar functions in defense as PP cells in the bark. Callus from trees of varying resistance seem to reflect the relative resistance of the trees from which they are derived, and this study indicates that some mechanisms of resistance can be studied using callus from trees of different resistance.     

Expression profiling of the lignin biosynthetic pathway in Norway spruce using EST sequencing and real-time RT-PCR

Lignin biosynthesis is a major carbon sink in gymnosperms and woody angiosperms. Many of the enzymes involved are encoded for by several genes, some of which are also related to the biosynthesis of other phenylpropanoids. In this study, we aimed at the identification of those gene family members that are responsible for developmental lignification in Norway spruce (Picea abies (L.) Karst.). Gene expression across the whole lignin biosynthetic pathway was profiled using EST sequencing and quantitative real-time RT-PCR. Stress-induced lignification during bending stress and Heterobasidion annosum infection was also studied. Altogether 7,189 ESTs were sequenced from a lignin forming tissue culture and developing xylem of spruce, and clustered into 3,831 unigenes. Several paralogous genes were found for both monolignol biosynthetic and polymerisation-related enzymes. Real-time RT-PCR results highlighted the set of monolignol biosynthetic genes that are likely to be responsible for developmental lignification in Norway spruce. Potential genes for monolignol polymerisation were also identified. In compression wood, mostly the same monolignol biosynthetic gene set was expressed, but peroxidase expression differed from the vertically grown control. Pathogen infection in phloem resulted in a general up-regulation of the monolignol biosynthetic pathway, and in an induction of a few new gene family members. Based on the up-regulation under both pathogen attack and in compression wood, PaPAL2, PaPX2 and PaPX3 appeared to have a general stress-induced function. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Microchemical analysis of laser-microdissected stone cells of Norway spruce by cryogenic nuclear magnetic resonance spectroscopy

Stone cells (sclereids) in Norway spruce (Picea abies) bark have been reported to be highly lignified tissues that are important in physical defence against bark beetle invasion. Microchemical analyses of the low-molecular weight compounds in the stone cells of Norway spruce were carried out using laser microdissection in combination with cryogenic nuclear magnetic resonance and mass spectrometry (LMD/NMR/MS). Two phenolic compounds, the stilbene astringin and the dihydroflavonol dihydroxyquercetin 3′-O-β-d-glucopyranoside, were identified indicating that stone cells are more than just repositories for lignin. Both of these compounds were also found to be present in other phloem tissue at a higher level than in the stone cells based on quantification by cryogenic ¹H NMR. Our results suggest that stone cells may be involved in chemical as well as physical defense against bark beetles and their associated microorganisms. This paper reports on the identification of secondary plant metabolites from a single laser-microdissected population of plant cells offering a sensitive new way to determine the chemical profile of specific plant cell types with a high degree of precision.     

The response of Norway spruce seedlings to simulated acid mist

Four pot experiments are reported in which Norway spruce ( Picea abies (L.) Karst) seedlings, of different nutrient status, were treated with acid mist for one growing season in open-top chambers (OTCs). Combinations of H⁺, SO₄²⁻, NH₄⁺ and NO₃⁻ were applied at different frequencies of application and supplying different doses of S and N kg ha⁻¹. Plant growth, visible injury, frost hardiness and nutrient status were observed. These experiments were undertaken to improve our understanding of the interaction of environmental factors such as nutrition and mist-exposure frequency on seedling response to N and S deposition.
Both acidity (pH 2·7) and SO₄²⁻ ions were necessary to induce visible injury. Mist containing SO₄²⁻, H⁺ and to a lesser extent NH₄⁺ significantly reduced winter frost hardiness. Increasing the misting frequency, and to a lesser extent the overall dose, increased the likelihood of acid mist causing visible injury and reducing frost hardiness. Post-planting stress, low N status and needle juvenility increased the likelihood of acid mist causing visible injury. Increased plant vitality, adequate N status and growth rate reduced the likelihood of acid-mist-induced reductions in frost hardiness.
Principles underlying the responses of spruce seedlings treated in controlled conditions to acid mist are discussed.     

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.     

Structure of lignins in developing xylem of Norway spruce

The developing xylem in a Norway spruce (Picea abies) clone was investigated during a growth season and compared to lignin from sapwood of the same tree clone. Klason and acid-soluble lignin contents were determined as well as the carbohydrate monomer distribution and protein content. By analyzing lignin thioacidolysis products, it was shown that only guaiacyl units could be detected in the materials, and the relative amount of beta-O-4' bonds was assessed. Monomeric and selected dimeric lignin products were identified by mass spectrometry. The specimens were embedded and thin sections examined by microscopy to determine the state of cell differentiation in the samples. In the spring and early summer, growth was very rapid and the intention was to collect tissue in which exclusively the middle lamella/primary cell wall had begun to lignify. Combining data regarding Klason lignin, protein content and carbohydrate monomer distribution with microscopy, it was found that the developing xylem sample from mid-June contained lignin from exclusively middle lamella/primary wall. The Klason lignin content in the developing xylem during the growth season was 20%, 5% and 10% in April, June and August, respectively. Thioacidolysis showed that the lignin had more condensed structures than lignin from the reference Norway spruce clone wood. Mass spectrometry showed that the developing xylem specimens from June and August contained more lignin structures with end-groups than the reference sample. These results suggest that lignification in the cambial layer and early developing xylem may take place more in a bulk fashion during the summer.     

Phenolics of mycorrhizas and non-mycorrhizal roots of Norway spruce

The occurrence and amount of soluble and insoluble phenolics in mycorrhizal and non-mycorrhizal roots of Picea abies (L.) Karst, were investigated, p-Hydroxybenzoic acid glucoside, picein, piceatannol and its glucoside, isorhapontin, catechin and ferulic acid could be identified by high-performance liquid chromatography in mycorrhizas of Picea abies-Lactarius deterrimus and Picea abies-Laccaria amethystea. Both types were collected from axenic cultures and the latter also from a spruce stand. The same phenolics occurred in non-mycorrhizal short roots from sterile cultures. However, the amounts of p-hydroxybenzoic acid glucoside, picein, catechin and cell wall-bound ferulic acid were considerably reduced in mycorrhizas from axenic culture, whereas the hydroxystilbenes piceatannol, its glucoside and worhapontin were not significantly reduced. Pure mycelia of Laccaria amethystea (Bull.) Murr, and Lactarius deterrimus Gröger were also analysed for phenolic compounds. Both fungal species contained none of the identified phenolics. The results are discussed with respect to mycorrhization in different mycorrhizal types.We are grateful to the Deutsche Forschungsgemeinschaft (Schwerpunktprogramm Physiologie der Bäume) and the Fonds der Chemischen Industrie for financal support. We thank Dr. V. Wray (GBF, Braunschweig, FRG) for linguistic advice.     

Seasonal development of the photosynthetic performance of Norway spruce (Picea abies [L.] Karst.) under magnesium deficiency

In order to investigate the influence of different magnesium nutrition on photosynthesis, one hundred 6-year-old spruce trees derived from one clone were planted in October 1990 into a special out-door experimental construction, where they were cultivated in sand culture with an optimal supply of nutrients, except magnesium, via circulating nutrient solutions. Magnesium was added to the nutrient solutions in three different concentrations, varying from optimal to severe deficient supplies. During the first vegetative period in 1991, photosynthetic performance and carboxylation efficiency were measured under saturating light, controlled CO₂ conditions, optimal temperature and humidity, using a minicuvette system.During summer, the trees under moderate magnesium deficiency developed tip yellowing symptoms on older needles, while the youngest needles remained green with unchanged chlorophyll contents. Trees under severe magnesium deficiency showed yellowing symptoms on all needle age classes combined with decreased chlorophyll contents in the youngest needles as well. In comparison with the controls, the photosynthetic performance of the 1-year-old needles was significantly lower in both deficiency treatments. The same was observed in the youngest needles of the trees under severe deficiency. Trees under moderate deficiency treatment decreased in photosynthetic performance during the summer without reduction of chlorophyll contents. The reduction of photosynthetic rates corresponded to a decrease in carboxylation efficiency, which is taken as a measure of the activity of the enzyme ribulose-1,5-bisphosphate carboxylase. This reduction, together with the observed increase of carbohydrate contents in needles of trees growing under magnesium deficiency, led to the assumption that the photosynthetic carbonfixation is reduced as a consequence of the accumulation of carbohydrates.     

Growth response to different types of NPK-fertilizer in Norway spruce plantations in Western Denmark

In Norway spruce planted on former heathland and fertilized to increase production, a series of experiments (72 sample plots) was established throughout Central and Western Jutland (Denmark) during the springs of 1978 and 79. The sample plots were all fertilized with 120 kg nitrogen in each of two five-year periods. Different types of mixed nitrogen/potassim/phosphorus fertilizer were applied. According to results from fertilization trials in the 1950s and 60s a gain of 3–4 m³ · ha^-1 · year^-1 was expected. In contrast to these earlier findings, only 0, 76 m³ · ha^-1 · year^-1 was gained over the two periods as an average of all sample plots. The initial assumption that nitrogen is the main limiting factor for tree growth in Western Denmark no longer seems valid. The results might indicate that due to increasing nitrogen deposition during the 1970s and 80s, potassium and maybe phosphorus are developing into new minimum factors, limiting tree growth and devitalizing the forest ecosystem. Hence, fertilization on these soils should mainly be considered as a way to compensate such nutrient imbalances.     

The first crystal structures of a family 19 class IV chitinase: the enzyme from Norway spruce

Chitinases help plants defend themselves against fungal attack, and play roles in other processes, including development. The catalytic modules of most plant chitinases belong to glycoside hydrolase family 19. We report here x-ray structures of such a module from a Norway spruce enzyme, the first for any family 19 class IV chitinase. The bi-lobed structure has a wide cleft lined by conserved residues; the most interesting for catalysis are Glu113, the proton donor, and Glu122, believed to be a general base that activate a catalytic water molecule. Comparisons to class I and II enzymes show that loop deletions in the class IV proteins make the catalytic cleft shorter and wider; from modeling studies, it is predicted that only three N-acetylglucosamine-binding subsites exist in class IV. Further, the structural comparisons suggest that the family 19 enzymes become more closed on substrate binding. Attempts to solve the structure of the complete protein including the associated chitin-binding module failed, however, modeling studies based on close relatives indicate that the binding module recognizes at most three N-acetylglucosamine units. The combined results suggest that the class IV enzymes are optimized for shorter substrates than the class I and II enzymes, or alternatively, that they are better suited for action on substrates where only small regions of chitin chain are accessible. Intact spruce chitinase is shown to possess antifungal activity, which requires the binding module; removing this module had no effect on measured chitinase activity.     

Morphological and anatomical factors responsible for varied susceptibility of some species of spruce to infestation by spruce spider mie (Oligonychus ununguis Jacobi)

Differences in susceptibility to infestation by spruce spider mite of 3 investigated spruce species (Picea glauca ‘Conica’, P. pungens and P. omorika) may be due to features of anatomical and morphological structure of needles. In P. omorika, showing some resistance to the spruce spider mite, we noted lamellar structure of epidermal cell walls and extensive supporting tissues (hypodermis and sclerenchyma fibres) at the whole circumference of the needle.     

Fungal injury to seed tissues of Norway spruce,Picea abies (L.) Karst.

Norway spruce bore an abundance of cones in Finland in 2000, but these cones were often fungal-infected. The seeds had structural injuries that were revealed when seed samples were examined using light (LM) and a field emission scanning electron microscope (FESEM). Two main types of spores were found either in the tissues inside the seed coat or on the sarcotesta, the outermost layer of seed coat. The spores of Chrysomyxa pirolata appeared particularly in the nucellar tissue, where the cell walls were disintegrated at the middle lamellae and cytoplasm was disrupted. Degenerated remnants of fungal structures resembling aecial peridium were found close to aeciospores. The tissue of the megagametophyte differed also from that of a normal mature seed. Conidia of Thysanophora penicillioides were often encountered on the sarcotesta where the ordinary wax cover was missing. Fungal injury occurred in the nucellar layers that shelter the embryo and megagametophyte from desiccation and oxidation. Destruction of these structures together with rapid opening of the seed coat advance deterioration of seeds during storage and may cause unexpected economic losses in forest plant production.     

Growth and defence in young pine and spruce and the expression of resistance to a stem-feeding weevil

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.     

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.     

Root inoculation with a forest soil streptomycete leads to locally and systemically increased resistance against phytopathogens in Norway spruce

Soil streptomycetes are commonly antagonistic against plant pathogens. However, interactions involving increased defense responses in the host plant, leading to suppression of plant disease development, have not yet been detailed. Here, the mechanisms were studied of disease suppression by Streptomyces sp. GB 4-2 against Heterobasidion root and butt rot in Norway spruce (Picea abies) seedlings. GB 4-2 promoted mycelial growth of the phytopathogenic fungus, germination rate of fungal spores, extension of germ tubes and early colonization of outer cortical layers of the plant root. Reduced colonization of the inner cortical cell layers was accompanied by the induction of cell wall appositions, and increased xylem formation in the vascular cylinder emerged after bacterium-fungus coinoculation. Bacterial treatment led to decreased water content in roots and needles and increased photosynthetic yield (F(v)/F(m)) and peroxidase activities in needles. The infection of needles by Botrytis cinerea was reduced by bacterial pretreatment. Complex interactions of GB 4-2 with Norway spruce and Heterobasidion abietinum were discovered. The bacterium promoted the growth of the phytopathogenic fungus but induced plant defense responses. Host responses indicate that GB 4-2 induces both local and systemic defense responses in Norway spruce.     

Concentrations of nutritional and trace elements in needles of Norway spruce (Picea abies [L.] Karst.) as functions of the needle age class

The endogenous concentrations of the essential elements Ca, Cl, Cu, Fe, K, Mg, Mn, N, P and Zn, and of the nonessential elements Al, As, Ba, Br, Co, Cr, Cs, Hg, I, La, Na, Rb, Sb, Sc, Si and Sr were determined in 5 successive needle age classes. 40 mature spruce trees from 6 different sites were investigated individually. A given element usually shows smooth changes with the needle age class t. Trees on a given site usually have a similar dynamic behaviour. The same holds for the different site means. The concentrations can be approximated by functions c=f(t). Three different types of functions are required to describe the dynamic behaviour of 3 groups of elements that increase with t, and one for the elements that decrease with t. A given element usually can be described by the same type of function at all sites, even if its concentration differs widely. Exceptions are Mn, Co and Zn, which change from a decreasing function at low concentrations to an increasing function at high concentrations. Further irregulatities are found at some sites with Ca, Sr and Ba. These findings are corroborated by a multivariate statistical analysis.     

The mycorrhizal fungus Paxillus involutus transports magnesium to Norway spruce seedlings. Evidence from stable isotope labeling

Although it is well established that ectomycorrhizas improve the mineral nutrition of forest trees, there has been little evidence that they mediate uptake of divalent cations such as Mg. We grew nonmycorrhizal seedlings and seedlings mycorrhizal with Paxillus involutus Batsch in a sand culture system with two compartments separated by a 45-μm Nylon mesh. Hyphae, but not roots, can penetrate this net. Labeling the compartment only accessible to hyphae with ²⁵Mg showed that hyphae of the ectomycorrhizal fungus Paxillus involutus transported Mg to their host plant. No label was found in nonmycorrhizal control plants. Our data support the idea that ectomycorrhizas are important for the Mg nutrition of forest trees. This revised version was published online in June 2006 with corrections to the Cover Date.     

A geostatistical model to describe root vitality and ectomycorrhization in Norway spruce

Abstract

The spatial distribution of vital root tips and ectomycorrhizal (ECM) communities in forest soils is characterized by patchiness at a microscale level, mostly related to the distribution patterns of biotic and abiotic factors. A geostatistical model was applied to verify if spatial analyses could be useful in identifying an appropriate sampling method to study root tip vitality, ectomycorrhization and the ECM community. Root samples were collected from two high mountain Norway spruce forests (Trentino province, Italy) following a geometrical design. Laboratory microscopic and geostatistical ordinary kriging analyses were used to map tip vitality and ectomycorrhization degree, ECM richness and distribution grouped in “exploration types” (amount of emanating hyphae or presence and differentiation of rhizomorphs). Spatial gradients of the examined features existed at plant level, associated to the up-downslope direction (root tip vitality and ectomycorrhization, ECM richness) and distance from the stem base (ECM exploration types). The effectiveness of the geostatistical model used demonstrates that a geometrical sampling design, associated to spatial mapping techniques, can be useful in research where the tree, and not the forest, is the subject (mycological and phytopathological studies).