Variation in telomeric repeats of Scots pine (Pinus sylvestris L.)

The shortening of telomeres, the specific structures at the end of the eukaryotic chromosomes, has been associated with ageing and loss of cell replication or regeneration capacity. The aim of this study was to determine the variation in telomeric repeats during the life-span of Scots pine (Pinus sylvestris L.) by studying different tissues and age groups ranging from immature embryos to cambium, buds, and needles of mature, 200-year-old trees. The telomeric repeats in Scots pine including interstitial and centromeric repeats ranged from 0.9 up to 25 kb, and true telomeres were evaluated to have a mean length of 19.3 kb (±SE 0.17). Telomeres were observed to shorten with increasing tissue differentiation, embryonal samples having the longest repeats with an average length of 21.1 (±0.34) to 21.7 kb (±0.42) and the needles having, on average, the shortest repeats of 18.1 kb (±0.24). After germination, ageing per se had no significant effect on the length of telomeric repeats in cambium, bud, or needle tissues. In the older trees (50–200 years of age), the telomeres in stem cambium showed shortening towards the tree top. This is the first observation on such a position-related variation in telomeric repeats. Finally, there was a remarkable genotypic variation in the length of telomeric repeats, and this was consistent over the tissue types. In the small number of tested donor trees, the genotypic differences were not related to regeneration ability in tissue culture.     

Growth patterns in relation to drought-induced mortality at two Scots pine (Pinus sylvestris L.) sites in NE Iberian Peninsula

Drought-related tree mortality has become a widespread phenomenon. Scots pine (Pinus sylvestris L.) is a boreal species with high ecological amplitude that reaches its southwestern limit in the Iberian Peninsula. Thus, Iberian Scots pine populations are particularly good models to study the effects of the increase in aridity predicted by climate change models. A total of 78 living and 39 dead Scots pines trees were sampled at two sites located in the NE of the Iberian Peninsula, where recent mortality events have been recorded. Annual tree rings were used to (1) date dead trees; (2) investigate if there was an association between the occurrence of tree death and severe drought periods characterized by exceptionally low ratios of summer precipitation to potential evapotranspiration (P/PET); and (3) to compare the growth patterns of trees that died with those of surviving ones. Mixed models were used to describe the relationships between tree growth (in terms of basal area increment, BAI, and the percentage of latewood, LW%) and climate variables. Our results showed a direct association between Scots pine mortality and severe drought periods characterized by low summer water availability. At the two sites, the growth patterns of dead trees were clearly distinguishable from those of the trees that survived. In particular, the BAI of dead trees was more sensitive to climate dryness (low P/PET_summer, high temperatures) and started to decline below the values of surviving neighbors 15–40 years before the time of death, implying a slow process of growth decline preceding mortality.     

Topochemical investigation of early stages of lignin modification within individual cell wall layers of Scots pine (Pinus sylvestris L.) sapwood infected by the brown-rot fungus Antrodia vaillantii (DC.: Fr.) Ryv.

The initiation and progress of wood degradation of Pinus sylvestris sapwood exposed to the brown-rot fungus Antrodia vaillantii was studied on a cellular level by scanning UV microspectrophotometry (UMSP 80, Zeiss, MSP 800 Spectralytics). This improved analytical technique enables direct imaging of lignin modification within individual cell wall layers. The topochemical analyses were supplemented by light and transmission electron microscopy (TEM) studies in order to characterize morphological changes during the first days of degradation. Small wood blocks (1.5 × 1.5 × 5 mm) of Scots pine (P. sylvestris) were exposed to fungal decay by A. vaillantii for 3, 7, 11, 16, and 22 days. No significant weight loss was determined in the initial decay periods within three up to 7 days. After three days of decay the topochemical investigation revealed that the lignin modification starts at the outermost part of the secondary wall layer, especially in the region of the latewood tracheids. During advanced degradation after exposure of 22 days, lignin modification occurs non-homogeneously throughout the tissue. Even among the significantly damaged cells, some apparently unmodified cells still exist. Knowledge about lignin modification at initial stages of wood degradation is of fundamental importance to provide more information on the progress of brown-rot decay.     

Nitrogen dynamics in humus and soil beneath Sitka spruce (Picea sitchensis (Bong.) Carr.) planted in pure stands and in mixture with Scots pine (Pinus sylvestris L.)

Sitka spruce planted on nutrient-poor soils in mixture with pine or larch, unlike pure spruce, does not become N deficient and does not require N fertilizer. To test the hypothesis that N availability in the soil is enhanced beneath mixed species, the seasonal changes in different N forms were compared in humus (L+F+H) and soil beneath 15-year-old Sitka spruce (SS) and mixed Sitka spruce-Scots pine (SS and SP) planted on a gleyed heathland soil. Amounts of mineral and organic N extracted from humus in spring were significantly (p < 0.05) higher in SS and SP than in SS. Larger amounts were measured in the underlying soil, which favoured the deeper-rooting spruce and pine in SS and SP plots. Annual net N mineralization, measured by in-situ incubation, was 32 and 47 kg N ha^-1 in the surface 10 cm beneath SS and (SS and SP), respectively. In spring, readily mineralized organic N (waterlogged incubation at 30°C) was higher in humus and soil from (SS and SP) than from SS by 15 kg N ha^-1. The larger N pools beneath (SS and SP) were consistent with the higher total N content of the humus beneath (SS and SP), 446 compared with 255 kg N ha^-1 beneath SS. This indicated that beneath (SS and SP) N had been transferred from the underlying soil.     

The mycorrhizal community in a forest chronosequence of Sitka spruce [Picea sitchensis (Bong.) Carr.] in Northern England

Demography and fungal diversity of the belowground ectomycorrhizal community in a chronosequence of Sitka spruce [Picea sitchensis (Bong.) Carr.] in Northumberland, Northern England, were analysed; mycorrhizal root samples were taken from 6-, 12-, 30- and 40-year-old stands, and fungal fruiting bodies were collected in autumn to complement the survey. Naturally germinated seedlings less than 1 year of age (taken from the 30-year-old stand) were also examined. A total of 118,000 mycorrhizal root tips were extracted from 40 soil cores (ten per age class) and from the complete root systems of 25 seedlings and separated into active and senescent root tips according to their morphology and anatomy. Active tips were distinguished according to their mycobionts which were characterised and identified microscopically. Although almost 100% of all fine roots were mycorrhizal, EM fungal diversity throughout the chronosequence was low, consisting of a total of 16 species of which three were only found as fruiting bodies. Of the six mycobionts found most regularly below ground, Tylospora fibrillosa was the most common, colonising about 70% of all root tips and more than 90% of those of seedlings and young trees. Root density and mycorrhizal diversity increased, but percentage of vital root tips decreased with increasing tree age, levelling off in the 30- and 40-year-old stand. Among the five subdominant fungal species, Dermocybe crocea was found to have its peak of distribution in the 12-year-old stand and Russula emetica, Lactarius rufus, Hymenoscyphus ericae agg. and the unidentified Piceirhiza sulfo-incrustata in the 30- and 40-year-old stands. The possible correlations between the mycorrhizal community structure and biotic and abiotic factors are discussed.     

The influence of culture vessel head-space volatiles on somatic embryo maturation in Sitka spruce [Picea sitchensis (Bong.) Carr.]

The maturation of somatic embryos of Sitka spruce [Picea sitchensis (Bong.) Carr.] was found to be highly dependent on the method used to seal plastic Petri dishes. Large numbers of well-formed mature embryos developed if dishes were sealed with PVC cling-film (CF) whilst sealing with Parafilm M (PF) greatly reduced the numbers of embryos forming. Inclusion of potassium permanganate oxidation traps, normally used to deplete the atmospheric ethylene, greatly stimulated somatic embryo maturation under PF sealing. Similarly, traps of adsorption agents (Tenax, activated charcoal or soft white paraffin), capable of removing volatiles from the culture vessel head-space, stimulated somatic embryo maturation under PF sealing although to a lesser extent than the oxidation traps. Incorporation of silver nitrate or 2-chloroethylphosphonic acid (ethephon) in the culture medium indicated that ethylene was not the agent supressing somatic embryo maturation under PF sealing.Abbreviations ABA abscisic acid - CF PVC cling-film - PF Parafilm M     

Transpiration and canopy conductance in an inner alpine Scots pine (Pinus sylvestris L.) forest

Canopy transpiration (E_c) of a 150-year-old Pinus sylvestris L. stand in an inner Alpine dry valley, Tyrol, Austria was estimated throughout two growing seasons 2011 and 2012 by means of xylem sap flow measurements. Although there were prolonged periods of limited soil water availability, E_c did not show a clear trend with respect to soil water availability and averaged 0.4 ± 0.19 mm day⁻¹ under conditions of non-limiting soil water availability and 0.37 ± 0.17 mm day⁻¹ when soil water availability was limited. This is because canopy conductance declined significantly with increasing evaporative demand and thus significantly reduced tree water loss. The growing season total of E_c was 74 mm and 88 mm in 2011 and 2012, respectively, which is significantly below the values estimated for other P. sylvestris forest ecosystems in Central Europe, and thus reflecting a strong adaptation to soil drought during periods of high evaporative demands.     

Cytokinin pulse-mediated shoot organogenesis from cotyledons of Sitka spruce [Picea sitchensis (Bong.) Carr.] and high frequency in vitro rooting of shoots

Shoot formation from cotyledons of Sitka spruce was induced by a 2 h incubation in an aqueous solution of 400 μM benzyladenine, followed by culture on medium lacking plant growth regulators. Several basal media were assessed. Murashige and Skoog (1962) based medium was found to be optimal on which, after 25 weeks, 62.5% of cotyledons produced shoots with a mean of 10.7 shoots per cotyledon. Shoots, detached from cotyledons and propagated individually by axillary bud proliferation, rooted with a frequency of 84% following culture in half-strength MS-based medium lacking growth regulators under low illumination (1.5 μmol m⁻²s⁻¹). Rooted plants were successfully transferred to ex vitro conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of stand age on greenhouse gas fluxes from a Sitka spruce [Picea sitchensis (Bong.) Carr.] chronosequence on a peaty gley soil

The influence of forest stand age in a Picea sitchensis plantation on (1) soil fluxes of three greenhouse gases (GHGs – CO₂, CH₄ and N₂O) and (2) overall net ecosystem global warming potential (GWP), was investigated in a 2‐year study. The objective was to isolate the effect of forest stand age on soil edaphic characteristics (temperature, water table and volumetric moisture) and the consequent influence of these characteristics on the GHG fluxes. Fluxes were measured in a chronosequence in Harwood, England, with sites comprising 30‐ and 20‐year‐old second rotation forest and a site clearfelled (CF) some 18 months before measurement. Adjoining unforested grassland (UN) acted as a control. Comparisons were made between flux data, soil temperature and moisture data and, at the 30‐year‐old and CF sites, eddy covariance data for net ecosystem carbon (C) exchange (NEE). The main findings were: firstly, integrated CO₂ efflux was the dominant influence on the GHG budget, contributing 93–94% of the total GHG flux across the chronosequence compared with 6–7% from CH₄ and N₂O combined. Secondly, there were clear links between the trends in edaphic factors as the forest matured, or after clearfelling, and the emission of GHGs. In the chronosequence sites, annual fluxes of CO₂ were lower at the 20‐year‐old (20y) site than at the 30‐year‐old (30y) and CF sites, with soil temperature the dominant control. CH₄ efflux was highest at the CF site, with peak flux 491±54.5 μg m⁻² h⁻¹ and maximum annual flux 18.0±1.1 kg CH₄ ha⁻¹ yr⁻¹. No consistent uptake of CH₄ was noted at any site. A linear relationship was found between log CH₄ flux and the closeness of the water table to the soil surface across all sites. N₂O efflux was highest in the 30y site, reaching 108±38.3 μg N₂O‐N m⁻² h⁻¹ (171 μg N₂O m⁻² h⁻¹) in midsummer and a maximum annual flux of 4.7±1.2 kg N₂O ha⁻¹ yr⁻¹ in 2001. Automatic chamber data showed a positive exponential relationship between N₂O flux and soil temperature at this site. The relationship between N₂O emission and soil volumetric moisture indicated an optimum moisture content for N₂O flux of 40–50% by volume. The relationship between C : N ratio data and integrated N₂O flux was consistent with a pattern previously noted across temperate and boreal forest soils.     

Inhibition of somatic embryo maturation in Sitka spruce [Picea sitchensis (Bong.) Carr.] by butylated hydroxytoluene,a volatile antioxidant released by parafilm

Head-space volatiles above embryogenicPicea sitchensis (Bong.) Carr. (Sitka spruce) tissues cultured in glass Petri dishes sealed with Parafilm M or cling-film, were captured on Tenax adsorption traps and analysed by gas chromatography / mass spectrometry. Each sealing system released a single major compound into the head-space; butylated hydroxytoluene from Parafilm M and 2-ethyl-1-hexanol from cling-film. After two weeks sealed under Parafilm M butylated hydroxytoluene accumulated to 1.1 g g^–1 FW in tissues and subsequent somatic embryo maturation was prevented. When butylated hydroxytoluene was supplied via the head-space (100 g/250 ml flask) 0.5 g g^–1 FW accumulated in tissues after two weeks and no somatic embryo maturation occurred. Potentially phytotoxic metabolites of butylated hydroxytoluene included a substituted stilbenequinone, butylated hydroxytoluene quinone methide and butylated hydroxytoluene dimer.     

The effects of culture medium rigidity on adventitious bud production and tissue vitrification in needle cultures of Sitka spruce [Picea sitchensis (Bong.) Carr.]

Adventitious bud formation on Sitka spruce [ Picca sitchensis (Bong.) Carr.] needle explants was strongly dependent upon the rigidity of the culture medium. In general, of organogenesis was greatest on weak gels and poorest on more rigid gels resulting from increased medium pH or agar strength. There was a significant interaction between agar strength and pH, with the optimum pH for organogenesis declining with increasing agar strength. Poor organogenesis at high agar concentrations was not due to toxic impurities since increased adventitious bud production could be stimulated by decreasing the medium pH whilst maintaining a high agar strength and an agar washing treatment had no significant effect. Although high levels of organogenesis could be sustained on weak gels the resultant adventitious shoots often showed severe vitrification. The frequency of shoots showing vitrification could be reduced by growing the tissues on harder media but this resulted in reduced shoot elongation. Vitrification of needle tissues did not stimulate the formation of adventitious buds in the absence of cytokinins.     

A tree of many faces: Why are there different crown types in Norway spruce (Picea abies [L.] Karst.)?

Norway spruce (Picea abies [L.] Karst.) has a pronounced ability to create different crown types embracing strongly hypotonic, epi-hypotonic, strongly amphitonic types as well as respective intermediate ones. Data of Holzer and Schultze (1987) were reanalyzed in order to identify major environmental components that may shape ecotypes and contribute to Gruber's (1989) hypothesis that phenotypic plasticity is different among different crown types. Environmental variables and crown types were assessed by Principal Components Analysis. The first principal component explained 74% of the variation that was mainly loaded by different temperature variables and altitude while the second principal component explained additional 23% mainly loaded by precipitation variables. Orientation had a statistically significant but small effect. Covariance analysis demonstrated that age had modified crown type in a way that more hypotonic types were phenotypically more variable. Overlaps between crown-type distributions were evaluated by Schoener's Index, which may range from ‘zero’ (no ecotypic overlap) to ‘one’ (complete ecotypic overlap). In the present paper this index resulted in pairwise values varying from 0.21 to 0.86. The ecotypic overlap matrix was symmetric, i.e. ecotypic pairs increased gradually with stepwise crown-type graduation. We discussed the adaptation strategy of Norway spruce based on our results and propose that adaptation in this species regarding crown architecture is mainly caused by adaptive differentiation in higher altitudes while in lower elevations phenotypic plasticity is the dominating factor.     

A branch bag and CO2 control system for long-term CO2 enrichment of mature Sitka spruce [Picea sitchensis (Bong.) Carr.]

This paper describes the construction and performance of branch bags and a CO₂ control system used to fumigate branches of mature Sitka spruce trees with air enriched in CO₂ (700 μmolmol^-1). It contains some examples of results obtained using the system over the course of the first two growing seasons. The branch bags have run continuously for 2 years with very few problems. CO₂ concentrations were within 20 μmol mol^-1 of the target concentration for more than 90% of the time. Temperatures within the bags were slightly higher than ambient (1–2 °C) and this had some effect on phenology. Attenuation of quantum flux density (photosynthetically active radiation) was 10–15%. The branch bag system has enabled investigation into the effects of elevated CO₂ on mature tissue without the problems and expense of fumigating whole trees. Growth in elevated CO₂ resulted in an increase in starch and a decrease in soluble protein content of needles. Stomatal conductance was higher in elevated CO₂ grown needles, and there was some evidence of an increase in photosynthetic capacity.     

Uptake of ammonium and nitrate ions from acid mist applied to Sitka spruce [Picea sitchensis (Bong.) Carr.] grafts over the course of one growing season

Cloned Sitka spruce grafts were subjected to NH₄NO₃ mist (1 mm day^-1 equivalent) acidified with H₂SO₄, for 4 days a week, from April until November. Three N treatments at pH 5.0 used 0.01 mol m^{-3 15}NH₄ ¹⁵NH₃, 1.6 mol m^{-3 15}NH₄ NO₃ or 1.6 mol m^-3 NH₄ ¹⁵NO₃. At pH 2.5, the treatments were¹⁵NH₄ ¹⁵NO₃,¹⁵NH₄NO₃ and NH₄ ¹⁵NO₃ all at 1.6 mol m^-3. At the end of the growing season,¹⁵N was found in every part of the trees, even when NH₄NO₃ was supplied at 0.01 mol m^-3. It was shown that both and could enter needles to a similar degree and be transported to the roots. No differences between pH treatments or clones could be demonstrated. Entry of labelled N via the roots, in those trees with unprotected compost, was reflected in higher fine root¹⁵N content, but no change in shoot¹⁵N levels. Per cent incorporation expressed as labelled N as a percentage of total N, was generally at its highest in bark, and in older needles in all treatments, values ranging from 5 to 11%. When¹⁵N content was expressed as total content for each tree part on a per tree basis, 25–36% was found in current year needles, with a further 12% in current year bark. The most reproducible data was that for dry weight of tree parts as per cent of the whole tree, where proportions compared closely between treatments and the two clones used. The implications of the results for cuticular transport mechanisms, N storage and internal cycling are discussed.     

Genetic parameters of morphological and physiological characteristics of containerized white spruce (Picea glauca [Moench.] Voss) seedlings

The root systems of containerized seedlings must be sufficiently developed and have adequate root plug cohesion to permit handling and the planting of the seedlings with minimal root damage. Genetic variability in morphological and physiological seedling characteristics of 75 open-pollinated white spruce families was estimated to determine whether genetic selection for improved seedling root systems is possible. Seedlings were grown for 2 years under standard cultural practices in a forest nursery. Gas exchange measurements and seedling morphological characteristics (height, diameter, shoot and root dry mass, root to shoot ratio) were measured at the end of the two growing seasons whereas seedling mineral (N, P, and K) status was assessed at the end of the first growing season. Genetic parameters (heritabilities—h ²—and genetic correlations) were estimated for every seedling characteristic and a strong genetic control associated with a large genetic variation was observed at both family (0.20 ≤ h_f² h_f^2  ≤ 0.88) and individual (0.21 ≤ h_i² h_i^2  ≤ 0.97) levels. A single, late-season measurement of physiological characteristics did not reveal physiological basis for family variability in seedling root growth. Nevertheless, the family variation was large enough to permit genetic improvement of 2-year-old seedling juvenile morphological characteristics. Strong, positive genetic correlations enable us to foresee using root collar diameter as an effective method for indirectly selecting white spruce families with heavier root systems.     

Contrasting responses to environmental changes by pine (Pinus sylvestris L.) growing on peat and mineral soil: An example from a Polish Baltic bog

A comparison of the tree-ring width of pines growing in areas adjacent to a peat bog and on a Baltic raised bog dome suggests that cambium activity in each tree group is affected by different factors. The study was aimed at pinpointing effects of meteorological factors on two pine populations growing under different hydrological conditions. The study further sought to identify periods during which anthropogenic pressure affected the two populations. The pines growing on mineral soil were characterised by ring-width growth–climate responses typical of this part of Europe, whereas the tree-ring growth of the pines growing on the peat bog showed no unequivocal relationship with meteorological conditions. The tree-ring width growth dynamics of that population responded primarily to changes in the level of the groundwater table. These changes were associated with human activities over the last two centuries. The development of the tree stands on the peat bog and in its immediate vicinity was reconstructed using this assumption and available historical evidence. A banding drainage ditch dug around 1880 resulted in the drying of the peat bog, the introduction of pines, and the forestation of the bog. Digging of central ditches (in the 1960s) resulted in periodic desiccation of the bog surface and reduction of the tree-ring widths; similar responses were observed in the 1980s as a consequence of the deepening and opening of the drainage ditches. The recent years are characterised by decreasing ring-width growth as a result of the damming of drainage ditches. Damming resulted in an increase of the groundwater table level in the bog and in a partial inundation of tree roots. This partial inundation interfered with gas exchange and adversely affected the health status of the pines studied. Our results allow conclusions to be drawn regarding peat bog palaeohydrology and facilitate absolute dating of changes associated with anthropogenic transformations. In active nature protection, such data are of key importance for understanding the functioning of a peatland and for planning active conservation measures.     

7500 yr of mire-pool development and the history of Pinus sylvestris (L.) in Sub-Arctic coastal Norway

In coastal North Norway, mire areas and mire pools frequently exist, but their development and time of origin are poorly known. In order to investigate the development of a coastal mire pool and relate its changes to known climatic changes, a sediment sequence from the mire-pool Lillevardhaugvatnet (c. 0.05 ha large), was ¹⁴C-dated, investigated for loss on ignition and analysed for pollen and botanical macrofossils.The bottom of the sequence dates c. 7500 cal. BP. The site gradually developed from a swamp forest c. 6200 cal. BP via a more open dwarf-shrub phase to a pool c. 5000 cal. BP. The pool grew in depth as the result of continuous peat growth damming the water body. The water level of the pool was probably lowered by erosional drain c. 2700 cal. BP. Redeposited peat in the sequence occurred c. 2100 cal. BP and c. 1100 cal. BP. ¹⁴C-dates and pollen indicate that erosion and redeposition of the peat surrounding the mire pool is a normal process, connected with the expansion of the water body.The combination of macrofossils and pollen accumulation rates (PAR) of P. sylvestris indicates that in small lakes in coastal areas of North Norway, a PAR of 200–400 cm^− 2 yr^− 1 is sufficient for indicating local presence of P. sylvestris. P. sylvestris is represented with abundant macrofossils between 4800 and 2100 cal. BP. It is suggested that a marked P. sylvestris decrease about 2100 cal. BP may be a combined effect of human impact and climatic deterioration. A possible final termination of the P. sylvestris population about 1600 cal. BP may be considered human-made.     

Variation of stilbene glucosides in bark extracts obtained from roots and stumps of Norway spruce (Picea abies [L.] Karst.)

Acetone-soluble compounds found in different root zones and stumps of Norway spruce (Picea abies [L.] Karst), which were grown on either peatland or a mineral soil site, were studied. Samples from stumps and roots of different sizes and ages were collected a day after the trees were felled. The wood and bark of stumps and three zones of the roots were separated and extracted with acetone in an ultrasonic bath. Extracts were silylated and analysed by gas chromatography–mass spectrometry. The stilbene glucosides astringin and isorhapontin were major compounds in the spruce bark samples. The resveratrol glucoside piceid and the flavonoid catechin were also extracted from spruce bark. We also found the lignan hydroxymatairesinol in some wood extracts. Total concentrations of stilbene glucosides in bark of stumps and different root zones varied between 0.53 and 8.29 % (w/w, dry weight) with isorhapontin being the major compound. Isorhapontin concentrations were highest in the spruce samples grown on mineral soil. The bark of the roots close to the stem is a rich source of stilbenes for commercial utilisation.