Genetic transformation of silver birch (Betula pendula) by particle bombardment

We used in vitro callus and shoot cultures as target material for genetic transformation of silver birch (Betula pendula Roth) by particle bombardment. Cultivation of in vitro shoot cultures before particle bombardment and a long selection period, combined with a high concentration of selective agent after bombardment, led to the production of transformed plantlets that were stable, and no escapes were found among the tree lines produced. Clonal variation in transformation efficiency was found in transient expression of the beta-glucuronidase gene in callus cultures and in plantlets transformed by stable integration of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) and neomycin phosphotransferase (npt2) genes.     

Analysis of EST-SSRs in silver birch(Betula pendula Roth.)

Simple sequence repeats(SSRs) defined as sequence repeat units between 1 and 6 bp occur abundantly in both coding and non-coding regions in eukaryotic genomes and these repeats can affect gene expression. In this study, ESTs(expressed sequence tags) of Betula pendula(silver birch) were analyzed for in silico mining of ESTSSRs, protein annotation, open reading frames(ORFs),designing primers, and identifying codon repetitions. In B.pendula, the frequency of ESTs containing SSRs was 7.8 %with an average of 1SSR/4. 78 kb of EST sequences. A total of 188 SSRs was identified by using MISA software and dinucleotide SSR motifs(65.9 %) were found to be the most abundant type of repeat motif followed by tri-(27.1 %),tetra-(4.8 %), and penta-(2.2 %) motifs. Based on ORF analysis, 175 of 178 sequences were predicted as ORFs and the most frequent SSRs were detected in 50 UTR(58.43 %),followed by in ORF(31.46 %) and in 30UTR(8.43 %). 102 of 178 ESTs were annotated as ribosomal protein, transport protein, membrane protein, carrier protein, binding protein,and transferase protein. For a total of 102 SSRs(57.3 %)with significant matches, a set of 102 primers(100 %) with forward and reverse strands was designed by using Primer 3 software. Serine(Ser, 19.6 %) was predominant in putative encoded amino acids and most of amino acids showed nonpolar(35.3 %) nature. Our data provide resources for B.pendula and can be useful for in silico comparative analyses of Betulaceae species, including SSR mining.     

Twilight far-red treatment advances leaf bud burst of silver birch (Betula pendula)

Bud development of boreal trees in spring, once initiated, is driven by ambient air temperature, but the mechanism triggering bud development remains unclear. We determined if some aspect of the diurnal or seasonal light regime influences initiation of bud burst once the chilling requirement is met. We grew 3-year-old birch plantlets cloned from a mature tree of boreal origin in light conditions realistically simulating the lengthening days of spring at 60 degrees N. To emulate the reduction in red to far-red light (R:FR) ratio between daylight and twilight, one group of plantlets was subjected to reduced R:FR ratio in the morning and evening in addition to progressively lengthening days, whereas the other group was subjected to the same R:FR ratio throughout the day. The reduced R:FR ratio of twilight advanced bud burst by 4 days compared with the reference group (P = 0.04). To assess the interplay between the fulfillment of the chilling requirement and the subsequent response to warming, we fitted a thermal time model to the data with separate parameterizations for the starting dates of heat sum accumulation in each treatment. Least-squares fitting suggested that bud development started in light regimes corresponding to late March, almost two months after the chilling requirement for dormancy release was satisfied. Therefore, shortening night length or increasing day length, or both, appears to be the cue enabling bud development in spring, with twilight quality having an effect on the photoperiodic response. If twilight alone were the cue, the difference in bud burst dates between the experimental groups would have been greater than 4 days. The result gives experimental support for the use of thermal-time models in phenological modeling.     

Seasonal and within-stem variations of neutral lipids in silver birch (Betula pendula) wood

Neutral lipids were analyzed in stem wood of a 7-year-old clone and in five 35-70-year-old mature trees of silver birch (Betula pendula Roth). In young trees and in mature wood of old trees, the free fatty acid fraction comprised less than 5% of the concentration of triacylglycerols (TG). The concentration of free linoleic acid was lowest in March when the young trees were dormant and highest during midsummer and September. In mature trees, the TG concentration increased toward the pith, indicating that living parenchyma cells close to the pith have a large TG storage capacity. The TG concentration (mean 0.51 +/- 0.02% of wood dry mass) remained constant throughout the year in young trees, whereas the concentration of beta-sitosterol, the dominant free sterol (mean 82.5 +/- 0.4% of total free sterols), decreased during spring and early summer when the temperature gradually increased, and increased during autumn when the trees became dormant. In young trees, we detected a seasonal interconversion between the free and esterified forms of beta-sitosterol and campesterol, and within the steryl ester fraction between squalene and betulaprenol-7. The concentration of esterified sterols/isoprenoids was exceptionally high, especially in the inner regions of mature stem wood (mean 0.6 +/- 0.03% of wood dry mass). No heartwood formation was detected.     

Photosynthesis and canopy characteristics in genetically defined families of silver birch (Betula pendula)

Net photosynthetic rates (A) of leaves in upper and lower crown layers (A(upper) and A(lower)), leaf area index (LAI), mean tilt angle (MTA), several leaf characteristics, and volume growth were observed in fast- and slow-growing families of a 14-year-old full-sib and half-sib family progeny test of Betula pendula Roth. Each measure of net photosynthetic rate was calculated after correcting measured net photosynthesis for the effects of environmental variables. The differences in A(upper) and LAI among families were significant. The proportions of the total variance assigned to family for A(upper), A(lower) and LAI were 33.64, 28.93 and 54.99%, respectively. The mean A(upper) and LAI of the fast-growing families were significantly higher than those of the slow-growing families, whereas the mean A(lower) of the fast-growing families was significantly lower than that of the slow-growing families. There were also significant differences among families in leaf size, leaf shape, and the ratios leaf fresh weight/area and leaf dry weight/area. Between 27.55 and 54.55% of the total variance in these characteristics could be assigned to the family effect. Volume growth was positively correlated with A(upper) and LAI, but it was most strongly correlated with A(upper) x LAI.     

Exploring life growth patterns in birch (Betula pendula)

The conversion of homogeneous plantations of conifers to mixed or broadleaved forests is part of sustainable forest management strategies in Western Europe. For the conversion of pine plantations on sandy soils, birch seems a convenient choice. Yet, thus far, little is known about the growth of mature birches outside Northern Europe. We sampled 32 birch trees in forests throughout the sandy ecoregion in northern Belgium at sites that varied in soil and local stand conditions. Half of the birches showed the characteristic pattern in growth (expressed as basal area increment) with a sharply increasing growth till around the age of 25 years followed by a steep decrease in growth. The growth was strongly related to tree age and crown dimensions, only weakly related to soil conditions, and not related to the local stand conditions.     

Isolation of genes up-regulated by copper in a copper-tolerant birch (Betula pendula) clone

Suppression subtractive hybridization (SSH) was used to isolate genes differentially expressed following exposure to copper (Cu) in a naturally selected Cu-tolerant birch (Betula pendula Roth.) clone originating from a disused lead/zinc smelter. Of the 352 cDNA fragments initially isolated, 108 were up-regulated by Cu, of which 55 showed over twofold induction by macroarray analysis. Searches against protein databases (Blastx) and sequence analysis provided the tentative identity of 21 genes. Three fragments lacked homology to any sequences in the databases. Most of the identified genes are involved in cellular transport, regulation or cell rescue and defense. Several genes have not previously been reported to be up-regulated by Cu, e.g., plasma intrinsic protein 2, glutamine synthetase and multi-drug resistance-associated protein (MRP4). The expression of MRP4, a vacuolar sorting receptor-like protein and an unidentified gene was studied in more detail by quantitative real-time PCR. These genes showed stronger up-regulation by Cu in the roots and shoots of the Cu-tolerant birch clone compared with a less tolerant clone. Clear clonal differences in gene expression were observed, e.g., for the regulator of chromosome condensation family protein, DnaJ protein homolog, vacuolar sorting receptor-like protein and MRP4. These findings contribute to our understanding of Cu tolerance in birch, a pioneer plant in metal-contaminated soils.     

Effects of elevated concentrations of ozone and carbon dioxide on the electrical impedance of leaves of silver birch (Betula pendula) clones

Effects of elevated concentrations of tropospheric ozone ([O3]) and carbon dioxide ([CO2]) on leaves of two silver birch (Betula pendula Roth) clones were monitored for three growing seasons (1998, 1999, 2000) by means of electrical impedance spectroscopy (EIS). The field trial with open-top chambers (OTCs) was conducted on two clones (Clone 4 and Clone 80) with five treatments and four independent replicates. Treatments were: (1) outside control, (2) chamber control, (3) 2x ambient [O3], (4) 2x ambient [CO2] and (5) 2x ambient [CO2] + 2x ambient [O3]. Fumigations started in 1999 and continued in 2000. Measurements were made in 1998 before the fumigations and thereafter EIS was carried out four times in each season. The impedance spectra of about 10 leaves from each tree at each time were measured at 42 frequencies between 80 and 1 MHz. Leaf spectra were modeled by a distributed circuit element model (DCE) (one DCE in series with a resistor), which yields the extracellular and intracellular resistances, the relaxation time and the distribution coefficient of the relaxation time. The EIS properties of the leaves changed significantly during the growing season when new leaves were expanding. The clones differed in their EIS properties. Clone 4 had a significantly higher extracellular resistance and distribution coefficient than Clone 80. The clones responded similarly to the fumigation treatments. Differences between treatments emerged especially during the second fumigation season in 2000. Elevated [O3] reduced both the relaxation time and the extracellular resistance, indicating cell membrane damage. Elevated [CO2] increased the intracellular resistance, indicating changes in symplastic composition. The biological interpretation of the EIS parameters in birch leaves is discussed.     

Differential responses of silver birch (Betula pendula) ecotypes to short-day photoperiod and low temperature

We investigated interrelations of dormancy and freezing tolerance and the role of endogenous abscisic acid (ABA) in the development of silver birch (Betula pendula Roth) ecotypes in controlled environments. Short-day treatment induced growth cessation, bud set and dormancy development, as well as initiation of cold acclimation and an increase in freezing tolerance. Subsequent low temperature and short days (12-h photoperiod) resulted in a significant increase in freezing tolerance, whereas bud dormancy was gradually released. The concentration of ABA increased in response to short days and then remained high, but ABA concentrations fluctuated irregularly when the dormant plants were subsequently exposed to low temperature during short days. Although there was a parallel development of freezing tolerance and bud dormancy in response to short days, subsequent exposure to low temperature had opposite effects on these processes, enhancing freezing tolerance and releasing dormancy. Compared with the southern ecotype, the northern ecotype was more responsive to short days and low temperature, exhibiting earlier initiation of cold acclimation, growth cessation and an increase in ABA concentrations in short days, and higher freezing tolerance, faster dormancy release and greater alteration in ABA concentrations when subsequently exposed to low temperature during short days. The rates and extent of the increases in ABA concentration may be related to increases in freezing tolerance and dormancy development during short days, whereas the extent of the fluctuations in ABA concentration may play an important role in enhancing freezing tolerance and releasing dormancy during a subsequent exposure to low temperature during short days.     

Effects of long-term,elevated ultraviolet-B radiation on phytochemicals in the bark of silver birch (Betula pendula)

Long-term outdoor experiments were conducted to investigate the effects of elevated ultraviolet-B (UV-B, 280-320 nm) radiation on secondary metabolites (phenolics and terpenoids) and the main soluble sugars (sucrose, raffinose and glucose) in the bark of silver birch (Betula pendula Roth) saplings. Saplings were exposed to a constant 50% increase in erythemal UV irradiance (UV-B(CIE); based on the CIE (International Commission on Illumination) erythemal action spectrum) and a small increase in UV-A radiation (320-400 nm) for three growing seasons in an irradiation field in central Finland. Two control groups were used: saplings exposed to ambient radiation and saplings exposed to slightly increased UV-A radiation. Concentrations of sucrose, raffinose and glucose in bark were higher in UV-treated saplings than in saplings grown in ambient radiation, indicating that stem carbohydrate metabolism was changed by long-term elevated UV radiation. Saplings in the elevated UV-A + UV-B radiation treatment and the UV-A radiation control treatment had significantly increased concentrations of certain UV-absorbing phenolics, such as salidroside, 3,4'-dihydroxypropiophenone-3-glucoside, (+)-catechin and (-)-epicatechin compared with saplings in ambient radiation. In contrast, the radiation treatments had no effect on the non-UV-B-absorbing terpenoids, papyriferic acid and deacetylpapyriferic acid. We conclude that plant parts, in addition to leaves, accumulate specific phenolic UV-filters in response to UV radiation exposure.     

Monolignol oxidation by xylem peroxidase isoforms of Norway spruce (Picea abies) and silver birch (Betula pendula)

We partially purified peroxidase isoform fractions from xylem extracts of a gymnosperm, Norway spruce (Picea abies (L.) Karst.), and an angiosperm, silver birch (Betula pendula Roth.), to determine the participation of xylem-localized peroxidases in polymerization of different types of lignin in vivo. Several peroxidase fractions varying in isoelectric point values from acidic to basic were tested for their ability to catalyze the oxidation of the monolignols coniferyl alcohol, sinapyl alcohol and p-coumaryl alcohol in vitro. All of the xylem peroxidases extracted from Norway spruce and most of those from silver birch showed the highest rate of oxidation with coniferyl alcohol in the presence of hydrogen peroxide. The exception was an acidic peroxidase fraction (pI 3.60-3.65) from silver birch that exhibited higher oxidation activity for sinapyl alcohol than for coniferyl alcohol. For the xylem enzyme fractions extracted from silver birch, the ability to oxidize the artificial phenolic substrate syringaldazine coincided with high specific activity for sinapyl alcohol. Therefore, we conclude that the acidic, neutral and basic xylem peroxidases of Norway spruce all function in the synthesis of guaiacyl-type lignin, whereas in silver birch the acidic peroxidases preferentially oxidize sinapyl subunits. The latter provides a mechanism for synthesis of guaiacyl-syringyl lignin typical of tracheid cell walls in angiosperm trees.     

Genotypic variation in drought response of silver birch (Betula pendula): leaf water status and carbon gain

To assess genotypic variation in drought response of silver birch (Betula pendula Roth), we studied the plasticity of 16 physiological traits in response to a 12-14-week summer drought imposed on four clones in two consecutive years. In a common garden experiment, 1-year-old clonal trees from regions with low (550 mm year(-1)) to high rainfall (1270 mm year(-1)) were grown in 45-l pots, and leaf gas exchange parameters, leaf water potentials, leaf osmotic potentials and leaf carbon isotope signatures were repeatedly measured. There were no clonal differences in leaf water potential, but stomatal conductance (gs), net photosynthesis at ambient carbon dioxide concentration, photosynthetic water-use efficiency, leaf carbon isotope composition (delta13C) and leaf osmotic potentials at saturation (Pi0) and at incipient plasmolysis (Pip) were markedly influenced by genotype, especially gs and osmotic adjustment. Genotypes of low-rainfall origin displayed larger osmotic adjustment than genotypes of high-rainfall origin, although their Pi0 and Pip values were similar or higher with ample water supply. Genotypes of low-rainfall origin had higher gs than genotypes of high-rainfall origin under both ample and limited water supply, indicating a higher water consumption that might increase competitiveness in drought-prone habitats. Although most parameters tested were significantly influenced by genotype and treatment, the genotype x treatment interactions were not significant. The genotypes differed in plasticity of the tested parameters and in their apparent adaptation to drought; however, among genotypes, physiological plasticity and drought adaptation were not related to each other. Reduction of gs was the first and most plastic response to drought in all genotypes, and allowed the maintenance of high predawn leaf water potentials during the drought. None of the clones exhibited non-stomatal limitation of photosynthesis. Leaf gs, photosynthetic capacity, magnitude of osmotic adjustment and delta13C were all markedly lower in 2000 than in 1999, indicating root limitation in the containers in the second year.     

Discoloration and associated fungi in stems of silver birch (Betula pendula Roth.) following logging damage

Study aim was to investigate discoloration and associated fungi in 50‐ to 70‐year‐old stems of Betula pendula damaged by logging. Investigated wounds (on 70 stems) were inflicted 4–18 years previously, and all of them had discoloration with mean radial spread (±SD) 2.9 ± 2.9 cm and longitudinal spread 50.3 ± 24.0 cm. Both wound area and length had a strong impact on a total length of discoloration (r = 0.665 and r = 0.745, respectively; p < 0.001), but had no influence on its spread beyond wound margins (r = ?0.035 and r = ?0.079). There was no correlation between the age of an injury and spread of the discoloration neither in vertical (r = ?0.110) nor in radial (r = ?0.280) direction. Average wound closure rate was 1.2 ± 0.5 cm year ^?1. The difference between discoloration length in stems with open and occluded injuries was insignificant (t‐test, p = 0.769). The principal fungi isolated from the wounds were ascomycetes (Epicoccum, Cadophora, Neonectria, Alternaria spp.) that were able to cause discoloration of wood with little effect on its mechanical properties and capable only for limited spread inside a stem of a living tree. In conclusion, results of the present study demonstrate that the amount of discoloured wood beyond logging wounds in B. pendula is mainly restricted to the damaged portion of a stem and that the associated loss of wood production remains low during about 10 years since the damage.     

The effects of heavy thinning on stem quality and timber properties of silver birch (Betula pendula Roth)

A study was carried out into the effects of heavy thinning onstem quality and timber properties of silver birch (Betula pendulaRoth). Comparisons were made with lightly thinned and unthinnedbirch stands. Birch stands from the UK were also compared withsimilarly managed stands from Germany and Denmark. In general,trees from lightly thinned and unthinned stands had similarcharacteristics. Heavy thinning enhanced diameter incrementthrough greater ring width in comparison with the other treatments.The timber properties of basic density, grain angle and shrinkagecoefficients in the radial, tangential and longitudinal directions,however, were largely unaffected. More and bigger branches andgreater stem taper were found on trees from heavily thinnedstands, but these stems still met the current Finnish veneerbirch quality and dimension standards. Comparisons between birchstands, managed by heavy and light thinning, from the UK andGermany and Denmark revealed no meaningful differences in timberproperties. The results suggest that veneer dimension and qualitylogs could be produced in the UK in rotations as short as 40years on good sites if heavy thinning is adopted.     

Variations in phenology and growth of European white birch (Betula pendula) clones

Phenology can have a profound effect on growth and climatic adaptability of northern tree species. Although the large interannual variations in dates of bud burst and growth termination have been widely discussed, little is known about the genotypic and spatial variations in phenology and how these sources of variation are related to temporal variation. We measured bud burst of eight white birch (Betula pendula Roth) clones in two field experiments daily over 6 years, and determined the termination of growth for the same clones over 2 years. We also measured yearly height growth. We found considerable genetic variation in phenological characteristics among the birch clones. There was large interannual variation in the date of bud burst and especially in the termination of growth, indicating that, in addition to genetic effects, environmental factors have a strong influence on both bud burst and growth termination. Height growth was correlated with timing of growth termination, length of growth period and bud burst, but the relationships were weak and varied among years. We accurately predicted the date of bud burst from the temperature accumulation after January 1, and base temperatures between +2 and -1 degrees C. There was large clonal variation in the duration of bud burst. Interannual variation in bud burst may have important consequences for insect herbivory of birches.     

Origin of the acetylated structures present in white birch (Betula pendula Roth) milled wood lignin

The occurrence and nature of acetate groups in the milled wood lignin (MWL) isolated from birch (Betula pendula Roth) has been addressed by spectroscopic (2D-NMR) and chemical degradative (derivatization followed by reductive cleavage, DFRC) methods. Considerable amounts of acetate groups were present in the MWL preparation. However, 2D-NMR analysis indicated that the lignin polymer is not extensively acetylated and that the major part of the acetate groups is attached to the xylan moieties present in the MWL preparation. Nevertheless, evidence of the presence of minor acetylation of the γ-carbon of the lignin side chain (<3% of both syringyl and guaiacyl lignin units) was provided by DFRC analysis.     

Browsing by large herbivores on Scots pine (Pinus sylvestris) seedlings in mixture with ash (Fraxinus excelsior) or silver birch (Betula pendula)

Abstract

Food selection by large herbivores occurs at a hierarchy of scales, for example landscape, patch or plant. Several hypotheses regarding food plant selection on patch or plant level have been developed. In this cafeteria-type design field experiment, conducted during one winter immediately after planting, we tested the effect of species mixture on browsing by large herbivores (mainly roe deer) on Scots pine seedlings in mixture with seedlings of ash (highly preferred) or silver birch (less preferred). Browsing on Scots pine was not affected by species mixture, neither in terms of the number of browsed pines nor browsing intensity. Instead, browsed biomass was positively and significantly correlated to the total biomass available for browsing. Also, there were differences due to species, with ash being most browsed (44.6%), followed by Scots pine (18.9%) and silver birch (11.6%). Browsed biomass per browsed seedling, however, was largest for Scots pine. In addition, browsed seedlings were initially taller compared to unbrowsed seedlings for all species. The main management implication in this study is that the species mixture did not influence large herbivore browsing on Scots pine seedlings. Hence, removing or discouraging more (or less) attractive browse species in early stages of pine regeneration activities seems unnecessary from the point of large herbivore browsing.     

Interactive effects of elevated ozone and temperature on carbon allocation of silver birch (Betula pendula) genotypes in an open-air field exposure

In the present experiment, the single and combined effects of elevated temperature and ozone (O(3)) on four silver birch genotypes (gt12, gt14, gt15 and gt25) were studied in an open-air field exposure design. Above- and below-ground biomass accumulation, stem growth and soil respiration were measured in 2008. In addition, a (13)C-labelling experiment was conducted with gt15 trees. After the second exposure season, elevated temperature increased silver birch above- and below-ground growth and soil respiration rates. However, some of these variables showed that the temperature effect was modified by tree genotype and prevailing O(3) level. For instance, in gt14 soil respiration was increased in elevated temperature alone (T) and in elevated O(3) and elevated temperature in combination (O(3) + T) treatments, but in other genotypes O(3) either partly (gt12) or totally nullified (gt25) temperature effects on soil respiration, or acted synergistically with temperature (gt15). Before leaf abscission, all genotypes had the largest leaf biomass in T and O(3) + T treatments, whereas at the end of the season temperature effects on leaf biomass depended on the prevailing O(3) level. Temperature increase thus delayed and O(3) accelerated leaf senescence, and in combination treatment O(3) reduced the temperature effect. Photosynthetic : non-photosynthetic tissue ratios (P : nP ratios) showed that elevated temperature increased foliage biomass relative to woody mass, particularly in gt14 and gt12, whereas O(3) and O(3) + T decreased it most clearly in gt25. O(3)-caused stem growth reductions were clearest in the fastest-growing gt14 and gt25, whereas mycorrhizal root growth and sporocarp production increased under O(3) in all genotypes. A labelling experiment showed that temperature increased tree total biomass and hence (13)C fixation in the foliage and roots and also label return was highest under elevated temperature. Ozone seemed to change tree (13)C allocation, as it decreased foliar (13)C excess amount, simultaneously increasing (13)C excess obtained from the soil. The present results suggest that warming has potential to increase silver birch growth and hence carbon (C) accumulation in tree biomass, but the final magnitude of this C sink strength is partly counteracted by temperature-induced increase in soil respiration rates and simultaneous O(3) stress. Silver birch populations' response to climate change will also largely depend on their genotype composition.