Histochemical and biochemical approaches to the study of phenolic compounds and peroxidases in needles of Norway spruce (Picea abies)

The three youngest age-classes of needles of Norway spruce ( Picea abies ) were collected from four sites in the Krusne Hory Mountains (Czech Republic) characterized by different levels of damage caused by environmental pollution. Histochemical methods did not reveal any differences in localization of phenolics among the needles. Mesophyll cells close to the epidermis of needles and cells around resin ducts and substomatal cavities often accumulated higher amounts of phenolics than the rest of the mesophyll cells, but this was independent of age and damage. Needles of different age- and damage-class did not show any marked changes in general lignification pattern. However, a lower intensity of histochemical detection of lignin was observed in needles from the most damaged site. This finding was confirmed by chemical analysis using thioglycolic acid. Generally, the amount of lignin in mesophyll cells was lower in damaged trees than in healthy ones. Using the Folin–Ciocalteau method, no significant differences in the total content of phenolics were observed in the needles, although HPLC revealed marked alterations in the forms of seven phenolic acids. Concentrations of conjugated forms of phenolic acids (esters and glycosides) were higher in damaged needles (255.9 μg g⁻¹ f. wt) than in healthy needles (189.8 μg g⁻¹ f. wt). By contrast, content of esterified phenolic acids incorporated into cell walls was higher in needles from healthy trees (101.1 μg g⁻¹ f. wt) than in damaged needles (78.3 μg g⁻¹ f. wt). Marked differences were also observed in the activity of soluble peroxidases, although the activity of ionically bound forms was approximately the same in healthy and damaged needles. The total amounts of chlorophylls and carotenoids decreased as environmental damage increased.     

Extracellular proteins in embryogenic suspension cultures of Norway spruce (Picea abies)

Embryogenic cell lines of Norway spruce ( Picea abies ) varying in growth habit and morphology were compared as regards profiles of extracellular proteins. Similar proteins were detected in the culture medium by SDS PAGE and in vivo labeling experiments, indicating that the proteins were secreted. Approximately 20 protein bands could be detected in the medium of each cell line. Three of the bands represented glycosylated proteins, as revealed by Concanavalin A staining. Some of the secreted proteins were similar for all tested embryogenic lines of Norway spruce, others were either specific for a group of cell lines or for individual cell lines. A correlation was observed between the morphology of the somatic embryos in a cell line and the presence of secreted proteins. The embryogenic cell lines of Norway spruce can be divided into two main groups. A and B, where A is characterized by somatic embryos with dense embryoheads and B by somatic embryos with loosely aggregated cells in their embryoheads. When proteins secreted from a cell line belonging to group A were added to cell lines belonging to group B, the somatic embryos of the B type developed further and became more similar in morphology to A-type embryos. These observations indicate that cell lines belonging to group A secrete certain proteins to the culture medium that are essential for the development of somatic embryos of Norway spruce.     

Ectomycorrhizal community structure varies among Norway spruce (Picea abies) clones

In northern boreal forests, the diversity of ectomycorrhizal (ECM) species is much greater than that of their host trees. This field study investigated the role of individual trees in shaping the ECM community. We compared ECM communities of eight Norway spruce (Picea abies) clones planted in a clear-cut area in 1994 with a randomized block design. In 2003, the ECM fungi were identified from randomly sampled root tips using denaturing gradient gel electrophoresis (DGGE) and rDNA internal transcribed spacer (ITS) sequence similarity. ECM diversity varied among clone groups, showing twofold growth differences. Moreover, according to detrended correspondence analysis (DCA), ECM community structure varied not only among but also within slow-growing or fast-growing clones. Results suggest that ECM diversity and community structure are related to the growth rate or size of the host. A direct or indirect influence of host genotype was also observed, and we therefore suggest that individual trees are partly responsible for the high diversity and patchy distribution of ECM communities in boreal forests.     

Importance of arabinogalactan proteins for the development of somatic embryos of Norway spruce (Picea abies)

The morphology of somatic embryos of Norway spruce ( Picea abies ) varies among different cell lines, from less developed somatic embryos with small embryonic regions (group B) to well developed embryos with large embryonic regions (group A). Only well developed somatic embryos will undergo a maturation process after a treatment with ABA and develop into mature somatic embryos, which is required for plant regeneration. We have previously shown that the presence of specific extracellular proteins can be correlated with the morphology of the somatic embryos. In the present study we show that extracellular proteins concentrated from group A cell lines can stimulate group B embryos to develop further and that seed extract can stably convert B embryos into A embryos. The arabinogalactan protein (AGP) fraction of the extracellular proteins and of the seed extract was shown to be an active component for stimulating B embryos to develop further. Furthermore, the amount and type of extracellular AGPs, as detected with β-glucosyl Yariv reagent and monoclonal antibodies, varied among different types of tissues and cell lines. The data show that development of somatic embryos in Norway spruce is associated with particular extracellular AGPs, which have a regulatory function.     

The rate of protein synthesis in needles of Norway spruce (Picea abies): Light stimulation, regulation through photophosphorylation, stress enhancement

The incorporation of ¹⁴C-leucine into the total-protein fraction of needles of Norway spruce (Picea abies [L.] Karst.) during short time incubation was used as a measure of protein synthesis in the light and in the dark. Light saturation curves, obtained for needles of different ages (new flush and 1 and 2 years old) or at different seasons (summer-winter) followed the Michaelis-Menten algorithm, exhibiting marked differences with regard to light saturation (V_max) and the half-saturation constant (K_{5. 2}). The light saturation curves of ATP level (mg g^?1 fresh weight) and of leucine incorporation into protein (nmol mg^?1 h^?1) matched each other, suggesting that photophosphorylation may be decisive for the rate of protein synthesis in the light. This is confirmed by the action spectrum of leucine incorporation. which resembled an action spectrum of leaf photosynthesis, and also by partial inhibition of protein synthesis by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of non-cyclic photophosphorylation. Light stimulated protein synthesis showed pronounced seasonal fluctuations with a summer maximum. Furmigation of 5 years old spruce trees for 3 months with SO₂ in combination with O₃ and/or NO₂ caused a distinct enhancement of the protein synthesis rate in the light and, at a reduced absolute level, also in the dark. A similar result was obtained for 40 to 70 years old spruce stands when healthy and sick trees were compared: the latter being afflicted by the novel type of forest decline, which is characterized by yellowish bronze discolouration of sun-exposed older needles and partial loss of older needle generations (3 to 4 years old). The 1 year old needles of the unhealthy trees showed a markedly increased ¹⁴C-leucine incorporation rate which, in the dark, was even more pronounced than in the light. Stress-physiological mechanisms, which could possibly explain this stimulation, are discussed.     

Biological activity, identification and quantification of gibberellins in seedlings of Norway spruce (Picea abies) grown under different photoperiods

Short photoperiod induces growth cessation in seedlings of Norway spruce ( Picea abies (L.] Karst.). Application of different gibberellins (GAS) to seedlings growing under a short photoperiod show that GA₉ and GA₂₀ can not induce growth. In contrast application of GA, and GA₄ induced shoot elongation. The results indicate that 3β-hydroxylation of GA₉ to GA₄ and of GA₂₀ to GA₁ is under photoperiodic control. To confirm that conclusion, both qualitative and quantitative analyses of endogenous GAs were performed. GA₁, GA₃, GA₄, GA₇, GA₉, GA₁₂, GA₁₅, GA₁₅, GA₂₀, GA₂₉, GA₃₄ and GA₅₁ were identified by combined gas chromatography-mass spectrometry in shoots of Norway spruce seedlings. The effect of photoperiod on GA levels was determined by using deuterated and ¹⁴C-labelled GAs as intermal standards. In short days, the amounts of GA₉, GA₄ and GA₁ are less than in plants grown in continuous light. There is no significant difference in the amounts of GA₃, GA₁₂, and GA₂₀ between the different photoperiods. The lack of accumulation of GA₉ and GA₂₀ under short days is discussed.     

Possible effect from shear stress on maturation of somatic embryos of Norway spruce (Picea abies)

Somatic embryogenesis is the only method with the potential for industrial scale clonal propagation of conifers. Implementation of the method has so far been hampered by the extensive manual labor required for development of the somatic embryos into plants. The utilization of bioreactors is limited since the somatic embryos will not mature and germinate under liquid culture conditions. The negative effect on mature embryo yields from liquid culture conditions has been previously described. We have described the negative effects of shear stress on the development of early stage somatic embryos (proembryogenic masses; PEMs) at shear stresses of 0.086 and 0.14 N/m². In the present study, additional flow rates were studied to determine the effects of shear stress at lower rates resembling shear stress in a suspension culture flask. The results showed that shear stress at 0.009, 0.014, and 0.029 N/m² inhibited the PEM expansions comparing with the control group without shear stress. This study also provides validation for the cross‐correlation method previously developed to show the effect of shear stress on early stage embryo suspensor cell formation and polarization. Furthermore, shear stress was shown to positively affect the uptake of water into the cells. The results indicate that the plasmolyzing effect from macromolecules added to liquid culture medium to stimulate maturation of the embryos are affected by liquid culture conditions and thus can affect the conversion of PEMs into mature somatic embryos. Bioeng. 2011; 108:1089–1099. © 2010 Wiley Periodicals, Inc.     

Storage protein accumulation during zygotic and somatic embryo development in Picea abies (Norway spruce)

Total protein was extracted from zygotic embryos and from somatic embryos of Picea abies (L.) Karst. (Norway spruce) cultured in vitro at different times during their development. An analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis of the protein extracts showed that protein composition and the temporal changes in protein abundance were very similar in the two embryo types. Both zygotic and somatic embryos accumulated storage proteins in abundance during their maturation phase of growth; the somatic embryos when cultured on medium containing 90 m M sucrose and 7.6 μ M ABA. The major storage proteins are composed of polypeptides with molecular masses of about 22, 28, 33 and 42 kDa and they are identical in both embryo types according to their molecular mass and average isoelectric points. These proteins are also the most abundant proteins in the female gametophytic tissue of the mature seed.     

Optimization of transient gene expression in pollen of Norway spruce (Picea abies) by particle acceleration

In order to optimize transient gene expression in Norway spruce pollen after DNA delivery with particle bombardment, effects of different conditions during homhardmenl were analysed using β-glucuroniduse (GUS) driven by the rice Act I promoter and Inciferase (LUS) driven by the tomato !at 52 promoter as reporter genes. Transient gene expression was significantly increased hy using two bombardments. Also the distance from the stopping plate to the sample was critical to gam maximum gene expression. There was no significant difference between gold and tungsten particles, and the number of positively stained pollen increased with increasing DNA concentration, from 5 to 40 pg DNA added in the DNA/tungsten solution The DNA delivery to Norway spruce pollen was most efficient at a chamber pressure above 70 kPa.     

NO2‐induced nitrate reductase activity in needles of Norway spruce (Picea abies) under laboratory and field conditions

The induction of activity of the enzyme nitrate reductase (NR, EC 1.6.6.1, 1.6.6.2) in needles of Norway spruce ( Picea abies [L.] Karst.) by nitrogen dioxide (NO₂) was studied under laboratory and field conditions. In fumigation chambers an increase in nitrate reductase activity (NRA) was detected 4 h after the start of the NO₂ treatment. During the first 2 days with 100 µg NO₂ m⁻³, NRA reached a constant level and did not change during the following 4 days. At the same level of NO₂, NRA was lower in needles from trees grown on NPK‐fertilized soil than on non‐fertilized soil. After the transfer of spruce trees from fertilized soil to NPK‐rich nutrient solution, NRA was transiently increased. This effect was assigned to root injuries causing nitrate transport to the shoot and subsequent induction of NRA. Neither trees on fertilized soil nor trees transferred to NPK‐poor nutrient solution had increased NRA unless NO₂ was provided. The NO₂ gradient in the vicinity of a highway was used to test the long‐term effect of elevated levels of NO₂ on needle NRA of potted and field‐grown spruce trees. Compared with less polluted sites, permanently increased NRAs were detected when NO₂ concentrations were above 20 µg m⁻³. Controls of field measurements some 10 years after the introduction of catalytic converters in cars showed no significant change neither in NO₂ levels nor in the decreasing NRA of spruce needles with the distance from the highway.     

Metabolism of nitrate and ammonium in seedlings of Norway spruce (Picea abies) measured by in vivo 14N and 15N NMR spectroscopy

In vivo ¹⁵N and ¹⁴N nuclear magnetic resonance spectroscopy was used to investigate the assimilation of nitrate and ammonium in seedlings of Norway spruce (Picea abies [L.] Karst.). The main objective was to study accumulation of free NH+₄ and examine to what extent the nitrogen source affects the composition of the free amino acid pools in roots, stems and needles. NH+₄ concentrations in plants growing in the presence of 0.5–50 mM ammonium were quantified using ¹⁴N NMR. The NH+₄ values in tissues ranged from 6 to 46 μmol (g fresh weight)^?1. with highest concentrations in roots and needles. The tissue NH+₄ peaked at 5.0 mM NH+₄ in the medium. and failed to increase when NH+₄ in the medium was increased to 50 mM, indicating metabolic control of the concentration of this cation in tissues. The ¹⁴N NMR spectra were used to estimate pH of the NH+₄ storage pools. Based on the pH sensitivity of the quintet of ¹⁴NH+₄ resonance, we suggest that the pH of the ammonium storage compartments in the roots and stems should be 3.7–3.8, and in needles 3.4–3.5, representing extremely low pH values of the tissue. ¹⁵N from nitrate or ammonium was first incorporated into the amide group of glutamine and then into α-amino groups, confirming that the glutamine synthetase/ glutamate synthase cycle is the major route of nitrogen assimilation into amino acids and thus plays a role in lowering the levels of NH+₄ in the cytoplasm. NH+₄ can also be assimilated in roots in plants growing in darkness. The main ¹⁵N-labelled amino acids were glutamine. arginine and alanine. Almost no ¹⁵N signals from needles were observed. Double labelling (δN + w, wN) of arginine is consistent with the operation of the ornithine cycle, and enrichment indicates that this cycle is a major sink of newly assimilated nitrogen. Nitrogen assimilation in roots in the presence of added methionine sulphoximine and glutamate indicated the catabolic action of glutamate dehydrogenase. The ¹⁵N NMR spectra of plants grown on ¹⁵N-urea showed a marked increase in the labelling of ammonium and glutamine. indicating high urease activity. Amino acids were also quantified using high pressure liquid chromatography. Arginine was found to be an important transport form of nitrogen in the stem.     

Embryology in Norway spruce (Picea abies). Immunochemical studies on transport of a seed storage protein

One of the main seed storage proteins of Norway spruce ( Picea abies ), is a salt-soluble protein with an average molecular mass of 42 kDa. This protein was localized by immunocytochemical methods in ultrathin sections of megagametophytes active in storage protein synthesis, as analyzed by SDS-PAGE. The megagametophyte in spruce starts accumulating storage materials, proteins and lipids, as the young embryo grows into the gametophytic tissue. It then continues to accumulate these storage products throughout seed development (Hakman 1993). Megagametophytes at an early stage of storage protein accumulation were chosen in this study for analysing the likely transport pathway of the proteins, since only a small amount of lipid had yet accumulated in the cells, and cell organelles were still easy to distinguish. An antibody against the 42 kDa storage protein showed very good reactivity with the 42 kDa protein in immunoblot experiments with total protein extracts from megagametophytes and embryos. In ultrathin sections of the megagametophyte, the antibodies were preferentially localized in the lumen of Golgi cisterna, in Golgi-associated vesicles, protein deposits close to the vacuolar membrane and in protein storage vacuoles (protein bodies). These observations indicate that the transport is mediated by the Golgi apparatus.
Also, proteins present in storage vacuoles in mature zygotic and somatic embryos showed intense labelling with these antibodies in ultrathin sections.     

Influence of cultivation method on root grafting in Norway spruce (Picea abies (L.) Karst.)

Root grafting is the process by which a functional union of two or more roots subsequent to their formation is formed. The above- and below-ground parts of three Norway spruce stands (natural stand, Umbric Luvisol; row-culture and group-culture, Planosol; stand ages 40, 43 and 43, respectively) of high site quality (I) were investigated. Stand densities were 1550, 1783 and 1722 stems ha^-1, respectively. In all investigated stands, root grafting was most sensitive to tree spacing. Grafts were observed in case the distance between the trees was 0.7–1.2 m. Grafts occurred always in areas of higher rooting density, in a row of the row-culture and within a tree group in the group-culture. Root grafting was enhanced in case of a narrower humus horizon in the group culture compared with the row-culture, 16.5 and 30 cm, respectively; the humus horizon contained 99% and 95% of conducting roots with d ≥ 5 mm, respectively. Root graftings occurred in 75% of excavated trees in the group-culture, in 37.5% of excavated trees in the row-culture and in 33.3% of excavated trees in the natural stand. Stand age was 24 years in the row-culture and 22 years in the group-culture at the beginning of root grafting. No grafts occurred between two suppressed trees, whereas in 86–100% of all cases, at least one tree was dominant or codominant. In row- and group-cultivated Norway spruce stands, the initial minimum diameter of the grafted root without bark was from 1 to 3 cm in 63% of cases. Grafting of roots with d < 1 cm or d > 10 cm was rare or absent. Root grafting had usually begun at the root age of 10–20 years (46% of cases). This revised version was published online in June 2006 with corrections to the Cover Date.     

Metabolism of tritiated and deuterated gibberellin A9 in Norway spruce (Picea abies) shoots during the period of cone-bud differentiation

A mixture of tritiated and deuterated gibberellin A₉ (GA₉) was injected into elongating shoots of Norway spruce [ Picea abies (L.) Karst.] grafts grown under environmental conditions that were either inductive (heat and drought, HD) or noninductive (cool and wet, CW) for flowering. The shoots were divided into needles and shoot stems. The metabolites were purified by high performance liquid chromatography (HPLC), detected by liquid scintillation counting of aliquots of collected fractions and identified by gas chromatography-mass spectrometry (GC-MS). Deuterated GA₉ was converted to deuterated GA₄ in both treatments. The major metabolite in the CW-treated material was GA₅₁. The HD-treated material did not convert GA₉ to GA₅₁, but a cellulase-hydrolysable GA₉-conjugate was formed. The same metabolites were found in the shoot stems, though in smaller amounts. The amounts of detected metabolites were higher in the HD material, caused by a higher rate of metabolism and/or smaller losses of the metabolites during sample purification. The estimated amounts of endogenous GAs show that the HD-treated material contained higher amounts of GA₉ but no differences in the amounts of GA₄ were found.     

Purification of intact chloroplasts from spruce (Picea abies) by free-flow electrophoresis

Highly purified chloroplasts were isolated from spruce ( Picea abies [L.] Karst.) needles by free-flow electrophoresis. The chloroplast crude suspension was separated into two structurally different populations. The two populations could not be distinguished according to their protein/chlorophyll ratios and protein patterns from two-dimensional gel electrophoresis. The O² evolution, however, showed differences: the chloroplast population deflected towards the cathode contained a major part of structurally intact chloroplasts in contrast to the population deflected towards the anode. The two populations were not contaminated by endoplasmatic membranes or mitochondria.     

Influence of UV-B radiation on photosynthetic activity and chlorophyll fluorescence kinetics in Norway spruce [Picea abies (L.) Karst.] seedlings

Norway spruce [Picea abies (L.) Karst.] seedlings were grown in greenhouses with two supplemental levels of ultraviolet-B (UV-B) radiation. Photochemical efficiency of photosystem II and vitality index were determined monthly. At the end of the experiment, growth, chlorophyll content and photosynthetic rates were measured. The data indicate that low temperature in winter affected light dependent processes in experimental plants including control, while the rise of ambient temperatures, moderate this effect. The synergistic effects of UV-B radiation and low temperatures could only be observed in the second winter period. Measurements of net photosynthetic activity in the second winter period showed significant differences between treated and untreated plants.     

Genomic organization of molecular differentiation in Norway spruce (Picea abies)

Diversity and differentiation among three populations representing the geographical domains commonly recognized within the natural distribution area of Picea abies were analysed by using a set of 292 AFLP (amplified fragment length polymorphism), SSR (single sequence repeat) and ESTP (expressed sequence tags polymorphism) markers. As usually observed in forest trees, results showed high within-population diversity (H(S) reaching 0.79) and low among-population differentiation (G(ST) approximately 2%). The genomic organization of differentiation was then investigated on the basis of a subsample of 150 AFLP, SSR and ESTP mapped markers. The number of the loci differentiating the Baltico-Nordic from the central European populations (25 loci) and, within the central European populations, the Alpine from the Hercyno-Carpathian populations (12 loci), were different. These 37 differentiated loci, with individual G(ST) values ranging from 0.008 to 0.20, were evenly distributed on all linkage groups and mostly followed the neutral expectations, suggesting genome-wide effects on differentiation. Nine of them however behave as 'outlier' loci indicating possible locus-specific selective effects. Contribution of ongoing evolutionary forces and historical effects to the geographical differentiation of the species are discussed.