Recovery of Norway spruce (Picea abies) seedlings from repeated drought as affected by boron nutrition

The effects of two boron (B) levels on growth, shoot water potential, gas exchange and nutrient accumulation in Norway spruce [Picea abies (L.) Karst.] seedlings were studied in a growth room experiment lasting 22 weeks which included well-watered control seedlings and seedlings exposed to one (8 days) or two (6+8 days) periods of drought and a rewatering period (8 days) at the end of the experiment. The effects of B and drought were monitored during drought and recovery. Needle B concentrations were 6 mg kg^–1 (–B treatment) and 34 mg kg^–1 (+B treatment) at the end of the experiment. The –B seedlings showed visible symptoms of damage in the upper shoot after repeated drought and had reduced height growth, root dry mass, allocation of biomass to roots and formation of root tips and mycorrhizas and reduced needle P, Ca, and Mg concentrations and contents. In contrast, ¹⁵N uptake, shoot water potential and gas exchange were not markedly affected by B. It can be concluded that the visible symptoms of damage at low B were probably related to reduced B transport due to repeated drought. In contrast, the effects of low B on growth, particularly of the roots, and on nutrient uptake can be regarded as early effects which occur before any influence on shoot water potential or gas exchange. The positive effects of B on root biomass and nutrient accumulation are of particular importance regarding the establishment of young seedlings in the field.     

Accumulation of ammonium in Norway spruce (Picea abies) seedlings measured by in vivo 14N-NMR

(14)N-NMR and (31)P-NMR have been used to monitor the in vivo pH in roots, stems, and needles from seedlings of Norway spruce, a typical ammonium-tolerant plant. The vacuolar and cytoplasmic pH measured by (31)P-NMR was found to be c. pH 4.8 and 7.0, respectively, with no significant difference between plants growing with ammonium or nitrate as the N-source. The (1)H-coupled (14) NH 4+ resonance is pH-sensitive: at alkaline pH it is a narrow singlet line and below pH 4 it is an increasing multiplet line with five signals. The pH values in ammonium-containing compartments measured by (14)N-NMR ranged from 3.7 to 3.9, notably lower than the estimated pH values of the P(i) pools. This suggests that, in seedlings of Norway spruce, ammonium is stored in vacuoles with low pH possibly to protect the seedlings against the toxic effects of ammonium ( NH 4+) or ammonia (NH3). It was also found that concentrations of malate were 3-6 times higher in stems than in roots and needles, with nitrate-grown plants containing more malate than plants grown with ammonium.     

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.     

Odors of Norway spruce (Picea abies L.) seedlings: differences due to age and chemotype

Small conifer seedlings (mini-seedlings) are less damaged by the large pine weevil Hylobius abietis (L.) (Coleoptera: Curculionidae) compared to conventional seedlings. Chemical difference between the seedling types is one possible explanation for this phenomenon. In the present paper, the emissions of volatile organic compounds (VOC) of 7- to 43-week-old Norway spruce [Picea abies (L.) Karst.] seedlings were analyzed. Collection and identification of the volatiles was made by solid phase micro-extraction and gas chromatography mass spectrometry (SPME–GC–MS). The enantiomers of α-pinene and limonene were separated in a two-dimensional GC (2D-GC). Most of the seedlings represented either a limonene- or a bornyl acetate-chemotype. Only minor changes in the volatile composition of the two types of seedlings were found during the first year. Age-related changes, however, were found in the volatiles released by wounded phloem where green leaf volatiles (GLVs) and borneol were the dominated VOC for young seedling. The attractive compound for the pine weevil, α-pinene, was first detected in the phloem emissions of 18- to 22-week-old seedlings. Different storage conditions of the seedlings during the winter/early spring-phase influenced the volatile composition in the phloem. High amount of GLVs was characteristic for the 43-week-old seedlings stored in naturally changing outdoor temperature, but not present in the seedlings winter-stored at a constant temperature of ?4 °C. The possible role of these observed differences in odor emissions between seedlings of different age and physiological status for the feeding preferences of the large pine weevil is discussed.     

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.     

Nitrogen distribution in young Norway spruce (Picea abies) trees as affected by pedospheric nitrogen supply

In numerous locations in Europe spruce trees are exposed to high loads of nitrogen. The present study was performed to characterize the distribution of nitrogen compounds under these conditions. For this purpose Norway spruce ( Picea abies [L.] Karst.) trees were cultivated under close-to-natural conditions of a forest understory in soil from an apparently nitrogen-limited field site in the Black Forest either with, or without supplementation of nitrogen as ammonium nitrate. After 11 and 20 months, growth, total nitrogen contents of the biomass, and total soluble non-proteinogenic nitrogen compounds (TSNN, i.e. nitrate, ammonium, soluble proteinogenic and non-proteinogenic amino compounds) in needles, xylem sap and phloem exudate were analysed. After 20 months of growth, N-fertilization had slightly enhanced the biomass of current-, but not of 1-year-old shoots. At both harvests, total N-content of 1-year-old needles was increased by N-fertilization, whereas current-year needles were not significantly affected. By contrast, TSNN was elevated by N-fertilization in both current-year and 1-year-old needles. The increase in TSNN was mainly attributed to an accumulation of arginine. Xylem sap analysis showed that the increase in TSNN of the needles was a consequence of enhanced nitrogen assimilation of the roots rather than the shoot. Since also TSNN in phloem exudates was enhanced, it appears that N-fertilization elevates the cycling pool of amino compounds in young Norway spruce trees. However, this pool seems to be subject to metabolic interconversion, since mainly glutamine and aspartate are transported in the xylem from the roots to the shoot, but arginine accumulated in the needles and the phloem.     

Freezing stress and membrane injury of Norway spruce (Picea abies) tissues

Effects of sub-zero temperatures (−5 to −35°C) on the tissues of needles, buds and shoots of Norway spruce [ Picea abies (L.) Karst.] were studied. The freezing caused increased efflux of cellular electrolytes. Freezing injury of the primordial shoots and 1-year-old shoots was the result of the spontaneous freezing of a deep supercooled cellular water. The crystallization injures the cellular membranes leading to the loss of semipermeability and to the drastic efflux of K⁺. In the needles there was no deep supercooling of water and two patterns of changes in the membranes, depending upon the range of the applied temperatures, could be distinguished. At 0 to – 25°C, which do not kill the cells, we observed a disturbance in the membrane semipermeability as monitored by electrolytes efflux within a few hours after thawing of the needles. At lower temperatures (−35°C) we observed irreversible loss of the membrane semipermeability, and death of the tissue. Those changes occurred 10 h after thawing and were probably caused by the released lytic enzymes and some toxic compounds, which acted on the cellular membranes.     

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.     

Developmental disorders in buds and needles of mature Norway spruce, Picea abies (L.) Karst., in relation to needle boron concentrations

Dieback of the terminal shoot and consequently bushy growth induced by boron deficiency have been reported widely throughout the world in several tree species. Recently, similar growth damage was documented in half of the young spruce stands in eastern Finland. To clarify the role of B deficiency, the light microscopic structure of emerging buds and of developing and previous-year needles of mature Norway spruce (Picea abies L. Karst.) from damaged (D stand), partly damaged (PD stand) and healthy (H stand) stands were analysed. The samples, on which needle nutrient concentrations were also determined, were taken seven times between early spring (April) and early winter (November). Cell death characterized by precipitation of the cell content, possibly due to the release of tannins after membrane rupture, was seen in the apex of emerging buds, and this led to fatal damage in about half of the buds in the trees from the D stand, where the needle B concentration was well below the deficiency level of 4–5 mg kg⁻¹. Furthermore, an increase in living cells that accumulated tannins in the vacuoles, which is a common stress and/or defense reaction, was found in the primordial shoots of buds and in the differentiating needles in the PD and D stands. The increase in the areas of the central cylinder and of the xylem found in the needles indicate structural plasticity during needle differentiation to drought. The time frame for bud emergence from late May up to mid-September means that an adequate B supply is necessary throughout the summer in order to avoid fatal bud damage and thus bushy growth of the trees.     

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

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

Effects of liming and boron fertilization on mycorrhizas of Picea abies

Effects of liming and B fertilization on Norway spruce [Picea abies (L.) Karst.] mycorrhizas were studied in factorial field experiments. The lime was applied twice, about 30 years and 12 years before sampling (2000 and 4000 kg ha^-1 dolomite). B was applied at the rate of 1.5 kg B ha^-1 two years before sampling.Boron fertilization doubled the number of root tips in the top 10 mm of the humus layer. The proportion of dead short root tips was increased from 10 % in control plots to 29 % in the limed plots. Numbers of dead root tips were increased when both lime and B were applied. The % of mycorrhizas with external mycelium was slightly increased and the % of Piloderma croceum Erikss. and Hjortst. was decreased by lime. In conclusion, adverse effects of lime on mycorrhizas were found, which were ameliorated by B fertilization, but lime-induced B deficiency alone was not the only reason for the effects of lime on root mortality.     

Fine root dynamics in a Norway spruce forest (Picea abies (L.) Karst) in eastern Sweden

The annual dynamics of live and dead fine roots for trees and the field layer species and live/dead ratios were investigated at a coniferous fern forest (Picea abies L. Karts) in Sweden. Our methods of estimating the average amount of fine roots involved the periodic sampling of fine roots in sequential cores on four sampling occasions. The highest live/dead ratio was found in the upper part of the humus layer for both tree and field-layer species and decreased with depth. Most tree fine roots on the four sampling occasions were found in the mineral soil horizon, where 86, 81, 85 and 89% of <1 mm and 89, 88, 89 and 92% of <2 mm diameter of the total amounts of live fine roots in the soil profile were found. The mean amounts of live fine roots of tree species for the total soil profile on the four sampling occasions was 317, 150, 139 and 248 g m^?2 for <1 mm and 410, 225, 224 and 351 g m^?2 for <2 mm diameter fine roots. The related amount of dead fine roots was 226, 321, 176 and 299 g m^?2 and 294, 424, 282 and 381 g m^?2, respectively. Average amounts of live and dead fine-roots and live/dead ratios from other Picea abies forest ecosystems were within the range of our estimates. The production of fine roots, <1 and <2 mm in diameter, estimated from the annual increments in live fine roots, was 207 and 303 g m^?2. The related accumulation of dead fine roots was 257 and 345 g m^?2, The turnover rate of tree fine roots <1 mm in diameter in the total soil profile amounted to 0.7 yr^?1 for live and 0.8 yr^?1 for dead fine roots. The related turnover rates for tree fine roots <2 mm were 0.4 yr^?1 and 0.7 yr^?1. Our data, although based on minimum estimates of the annual fluxes of live and dead fine roots, suggests a carbon flow to the forest soil from dead fine-roots even more substantial than from the needle litter fall. Fine-root data from several Picea abies forest ecosystems, suggest high turnover rates of both live and dead tree fine-roots.     

Volatile organic compounds emissions in Norway spruce (Picea abies) in response to temperature changes

Volatile organic compound (VOC) emissions from Norway spruce ( Picea abies ) saplings were monitored in response to a temperature ramp. Online measurements were made with a proton transfer reaction – mass spectrometer under controlled conditions, together with plant physiological variables. Masses corresponding to acetic acid and acetone were the most emitted VOCs. The emission rates of m137 (monoterpenes), m59 (acetone), m33 (methanol), m83 (hexanal, hexenals), m85 (hexanol) and m153 (methyl salicylate, MeSa) increased exponentially with temperature. The emission of m61 (acetic acid) and m45 (acetaldehyde), however, increased with temperature only until saturation around 30°C, closely following the pattern of transpiration rates. These results indicate that algorithms that use only incident irradiance and leaf temperature as drivers to predict VOC emission rates may be inadequate for VOCs with lower H, and consequently higher sensitivity to stomatal conductance.     

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.     

Winter-drought induced embolism in Norway spruce (Picea abies) at the Alpine timberline

At the timberline in the Central Alps, climatic conditions during winter frequently cause excessive drought stress (frost drought, 'Frosttrocknis'), which we hypothesized to induce cavitation in trees. We investigated the extent of winter-embolism in Norway spruce ( Picea abies (L.) Karst.) growing near the timberline and analysed adaptations in vulnerability and anatomy. We found conductivity losses of up to 100% at the highest elevation (2020 m) correlated with low water potentials down to − 4.0 MPa. Vulnerability thresholds (50% loss in conductivity) decreased from − 3.39 MPa at 800 m to − 3.88 MPa at 1600 m corresponding to a decrease in tracheid cross-sectional area as well as pit and pit pore diameters. These thresholds were lower than potentials measured in embolized twigs near the timberline at the sampling dates probably due to lower potentials and/or a role of freeze-thaw events earlier in winter. Data indicated refilling processes, which may be of particular relevance for trees at the timberline, since adaptations in drought-induced vulnerability failed to prevent winter-embolism.     

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.     

Effects of Growth Retardants on Norway Spruce (Picea abies)

Young seedlings of Picea abies Karst, grown in nutrient solution were treated with the growth retardants Amo-1618, B-995, and CCC. These were added to the nutrient medium. B-995 and CCC retarded root and shoot growth in the concentrations 100, 10, and 1 mg/l. Growth was almost entirely inhibited by 300 mg/l, obviously due to toxicity. The effects of Amo-1618 were similar but more varying. GA counteracted the effects of all the retardants on shoot growth, but not on root growth.