Hormone levels in the cambial region of intact Picea abies during the onset of cambial activity

Microdialysis probes were used to sample the cambial region of Picea abies stems during the cambial reactivation period. The concentrations of ethylene and zeatinriboside in each sample were measured by gas chromatography and enzyme-linked immuno-sorbent assay, respectively. The ethylene level increased throughout the experimental period, except for a temporary decrease that occurred about the time of budburst on the lower branches. The zeatin-riboside level changed relatively little during the same period. The results indicate that increased activity in the cambial region was associated with an increase in the level of ethylene but not of zeatinriboside.     

Ethylene effects on peroxidases and cell growth patterns in Picea abies hypocotyl cuttings

The effect of 2-chloroethylphosphonic acid (ethrel) on cell growth patterns and per-oxidase activity (EC 1.11.1.7) and location in young Norway spruce cuttings ( Picea abies [L.] Karst.) was investigated. The peroxidase activity in a fraction containing soluble and membrane bound enzymes show a diurnal variation, with decreased activity during the light period and a corresponding increase during the following dark period. The decrease during the day could to some extent be counteracted by treatment with ethrel. It appears that ethrel affects only peroxidases in the isolated membrane fraction, since peroxidases bound to the cell wall were not affected by ethrel. In vitro experiments indicated that the hydrophobicity of soluble peroxidases was increased by treatment with ethylene. Cytochemical localization of peroxidase activity in differentiating tracheids revealed a clear ethrel-induced increase in the tonoplast. It appears that ethylene affects soluble peroxidases in vivo in such a way that they are directed to a more hydrophobic environment, like the tonoplast. Treatment with ethrel also changed the appearance of the rough endoplasmic reticulum (ER) and Golgi apparatus. Dilated ER cisternae were observed on electron micrographs, as a result of treatment with ethrel. The number of vesicles produced by the Golgi apparatus and also the amount of vesicles fusing with the plasma membrane in secondary-wall-forming tracheids increased considerably. The results clearly indicate that the stimulatory effect of ethylene in spruce seedlings on lignification and cell wall formation, is due to a general stimulation on both synthesis, transport and secretion of cell wall material and not on a stimulation of peroxidase activity as reported for other species.     

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.     

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.     

Effect of local heating and cooling on cambial activity and cell differentiation in the stem of Norway spruce (Picea abies)

BACKGROUND AND AIMS The effect of heating and cooling on cambial activity and cell differentiation in part of the stem of Norway spruce (Picea abies) was investigated. METHODS: A heating experiment (23-25 degrees C) was carried out in spring, before normal reactivation of the cambium, and cooling (9-11 degrees C) at the height of cambial activity in summer. The cambium, xylem and phloem were investigated by means of light- and transmission electron microscopy and UV-microspectrophotometry in tissues sampled from living trees. KEY RESULTS: Localized heating for 10 d initiated cambial divisions on the phloem side and after 20 d also on the xylem side. In a control tree, regular cambial activity started after 30 d. In the heat-treated sample, up to 15 earlywood cells undergoing differentiation were found to be present. The response of the cambium to stem cooling was less pronounced, and no anatomical differences were detected between the control and cool-treated samples after 10 or 20 d. After 30 d, latewood started to form in the sample exposed to cooling. In addition, almost no radially expanding tracheids were observed and the cambium consisted of only five layers of cells. Low temperatures reduced cambial activity, as indicated by the decreased proportion of latewood. On the phloem side, no alterations were observed among cool-treated and non-treated samples. CONCLUSIONS: Heating and cooling can influence cambial activity and cell differentiation in Norway spruce. However, at the ultrastructural and topochemical levels, no changes were observed in the pattern of secondary cell-wall formation and lignification or in lignin structure, respectively.     

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.     

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.     

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.     

Micromorphological studies of adventitious bud formation on Picea abies embryos treated with cytokinin

Embryos of Picea abies (L.) Karst were pulse-treated with water or cytokinin for 2 h and then cultured on medium lacking cytokinin. Adventitious buds developed on cytokinin-treated embryos, but not on water-treated embryos. The general appearance and the surface morphology were similar on water and BA (benzyladenine)-treated embryos after 3 days. The epidermal cells were elongating after 6 days on water-treated embryos, while they were dividing on cytokinin-treated embryos. Furthermore, the cells surrounding the stomata had started to proliferate on BA-treated embryos. This was the first micromorphological sign of bud initiation. During the second week prominent meristemoids developed from these cells. A stoma was observed on the top of each meristemoid. The variation in developmental pattern of meristemoids among different embryos as well as within each embryo was small. However, during the subsequent development of bud primordia and buds, the morphological variation was significant. The meristemoids continued to develop into cone-shaped bud primordia, which successively changed shape during the transition to adventitious buds. The epidermal cells divided and the epidermis did not rupture during the formation of adventitious bud primordia. The epidermis was identified as the protoderm of the bud primordium.     

Ethylene accelerates the breakdown of cytokinins and thereby stimulates rooting in Norway spruce hypocotyl cuttings

Cuttings were taken from 4-week-old seedlings of Norway spruce ( Picea abies L. Karst.) raised at two different irradiation levels. Rooting experiments showed that root formation was increased by the ethylene formed by adding 1-aminocyclopropane-1-carboxylic acid ACC or Ethrel, especially in the slowly rooting cuttings grown under high light (HL). Cobaltousion. an ethylene synthesis inhibitor, delayed rooting, especially in the easily rooted cuttings grown under low light (LL).
Compounds isolated from the cuttings using immunoaffinity chromatography, on a column with antibodies against cytokinins, and separated by HPLC decreased in amount during the first week of the rooting period. An increase in ethylene production accelerated this process, especially in cuttings grown under HL, whereas cobaltous ion delayed it. We suggest that ethylene stimulates rooting by enhancing the degradation of cytokinins.     

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.     

Diagnosis of damage to Norway spruce (Picea abies) through biochemical criteria

Bioindication can be carried out at different hierarchical levels, eg. cell, organism, and ecosystem. While the monitoring of damage by visible criteria (e.g. loss of needles) is connected with the organism as a whole, the monitoring of damage by biochemical indicators is above all connected with cell metabolism.
The degree of vitality of a tree can be ascertained through the integration of a number of biochemical parameters. Furthermore, a differential diagnosis of a particular stress pattern can be carried out because of the feedback pattern of several biochemical indicators. In order to describe and interpret biochemical or physiological changes that have been caused by a number of factors, multivariate statistical methods are being used more frequently. Apart from cluster and discriminant analysis, it is especially factor analysis which provides a helpful tool when dealing with problems in the field of environmental analysis. Factor analysis can be used for an integrating as well as a differentiating assessment.
Within the framework of forest damage research, numerous changes at the level of cell metabolism have been detected to which a bioindicative character can be attached. A number of physiological and biochemical parameters with bioindicative character concerning Norway spruce are presented.     

High level histone H2A gene expression during early stages of adventitious bud formation in Norway spruce (Picea abies)

We have demonstrated the correlation between cell division and the expression of a histone H2A-encoding gene, His2A , in Norway spruce. Picea abies (L.) Karst and used a cDNA clone in in situ hybridization experiments to monitor the cytokinin-induced cell division during early stages of adventitious bud development. A general stimulation of division of epidermal and cortical cells followed upon the cytokinin treatment. After two weeks in culture a high mitotic activity was detected only in single cells or small groups of cells in the epidermis and subepidermal cell layers. These cells presumably constitute the early stages of meristem primordia. The small clusters of dividing cells enlarge and subsequently form adventitious buds. Cells of the meristem and needle primordia of adventitious buds divide frequently as do the corresponding cells in vegetative buds. A quiescent center is distinguished within the apical meristem of vegetative buds. These cells, in the summit of the domed meristem, divide with a considerably lower frequency than cells in the flanking region. Differences in the temporal expression pattern of the histone H2A gene in cells of the vascular tissue, detected between embryos germinating in vitro and bud-induced embryos, suggest that the cytokinin treatment affects the timing of cell divisions in the differentiating procambium.     

Segregation analysis of random amplified polymorphic DNA (RAPD) markers in Picea abies Karst.

The reliability of arbitrarily primed amplification products was tested. The segregation analysis of 266 amplification products obtained using 17 different 10-mer oligonucleotides in 34 megagametophytes from a single tree of Picea abies was carried out. Fifty-four out of the 165 variable bands fit the 1:1 segregation ratio expected for Mendelian traits. The segregation ratio of a subset of six RAPD markers in five other individuals from the same population confirmed their genetic nature. Our results strengthen the evidence previously reported that RAPDs markers can be considered Mendelian traits useful in the detection of genetic variability among both different individuals and populations.     

Movement and possible metabolism of ethylene in dormant Picea abies

Ethylene or ethane was added to and sampled from the base, middle and top of the stem of intact Picea abies (L.) Karst. using microdialysis probes. The endogenous concentrations of ethylene and ethane in all positions were below 5 × 10^–9 mol l^–1 throughout the experimental period. Applied ethylene or ethane was always detected in probes above the probe used for the application, never below. If increased ethylene or ethane concentrations were detected, the ethylene concentrations initially increased but thereafter decreased to the basal level, whereas levels of ethane steadily increased. It is proposed that the maintainance of low ethylene levels is important in the regulation of wood formation.     

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.