Plant regeneration from encapsulated somatic embryos of pedunculate oak (Quercus robur L.)

Summary Cotyledonary Quercus robur L. somatic embryos from two cell lines were encapsulated in 4% (w/v) sodium alginate. An artificial endosperm was provided by the addition of P₂₄ medium plus 3% (w/v) sucrose. Oak somatic embryos and oak synthetic seeds were germinated on P₂₄ medium plus 0.1 μM indole-3-butyric acid and 0.9 μM 6-benzylaminopurine or were dehydrated prior to germination. The highest conversion rates (26%) were obtained with encapsulated somatic embryos as well as artificial endosperm-coated somatic embryos. Encapsulation improved the regeneration into oak plantlets in one of the two cell lines tested. The artificial endosperm had no additional beneficial effect on conversion frequency, but increased germination rate in one cell line tested. Significant higher conversion could be attributed to slow desiccation compared to the non-encapsulated control. Cold storage as a post-maturation treatment had no influence on the germination ability of oak synthetic seeds. Differences in the response of the cell lines with respect to conversion frequencies and timing of germination were observed. Fifty-six well-developed plantlets regenerated 12 wk after germination, and 29 plants were transferred to the greenhouse, where they have been successfully established in substrate.     

Inheritance and molecular variations of PCR-SSCP fragments in pedunculate oak (Quercus robur L.)

Single-strand conformaiton polymorphism (SSCP) profiles of six PCR-amplified fragments (250–800 bp) were analyzed in three full-sib families of pedunculate oak (Quercus robur L.) and their parents. Among the six fragments, four were polymorphic and one exhibited complex patterns that were not changed by varying the SSCP conditions. The number of bands for the analyzed fragments varied between two and four among individuals regardless of fragment size. As shown by segregation data, the variation in the number of bands between trees could only be attributed to the allelic composition (homozygotes vs heterozygotes): a genotype that exhibited two bands was presumptively homozygous, wheras a genotype exhibiting three or four bands was heterozygous. Mendelian proportions were observed in all crosses for each polymorphic fragment. In one cross, we could clearly identify a null allele due to a possible mutation at a primer site. Single-base mutations and short insertion-deletions were shown to be the molecular causes of the SSCP polymorphism observed between different alleles. The use of SSCP as a technique to identify co-dominant markers of PCR fragments (up to 800 bp) is recommended for gene diversity studies or for gene mapping.     

Physiological and morphological characteristics during development of pedunculate oak (Quercus robur L.) zygotic embryos

The developmental stages of oak zygotic embryos (ZEs) are characterized here according to morphological and physiological features. Seeds were harvested from June to September in 1-week intervals. Excised embryos were classified into four stages of development by using growth parameters. For physiological characterization, endogenous levels of abscisic acid (ABA), indole-3-acetic acid (IAA), l-proline, starch content and water status were determined. The expression of the oak legumin storage protein gene was tested in immature cotyledonary ZEs before and after ABA treatment. The ABA levels of the embryos showed a significant peak during the intermediate stage of maturation (stage III) and then decreased again at the end of the late maturation phase (stage IV). Concomitant with ABA, the moisture content declined with the maximum embryo size. High IAA levels were found at the beginning of embryo enlargement as exponential growth occurred (stage II) but decreased during further development. Starch accumulated gradually in the course of maturation, whereas significant values were found in stage IV ZEs near shedding. Proline, on fresh weight basis, was high during stages I and II. Osmotic potential increased when, by rapid dry matter accumulation, stage II ZEs reached their maximum size during early intermediate development. Expression of precocious germination was higher on hormone-free medium, in particular, among stage II and stage III ZEs. Variations in phytohormone levels in combination with changes in tissue water status seem to be important factors for oak ZE development.     

Stem hydraulic conductance of European beech (Fagus sylvatica L.) and pedunculate oak (Quercus robur L.) grown in elevated CO2

Over two seasons in c. 600 ppm CO₂, oak had lower stomatal conductancein CO₂-enriched compared to amblent air. Beech showed no responseto CO₂ concentration on sunny days. Mirroring this pattern,exposure to elevated CO₂ reduced whole-shoot hydraulic conductanceper unit leaf area in oak, but not in beech. Key words: Climate change, Fagaceae, gas exchange, trees, water relations     

Transport of organic sulfur and nitrogen in the roots of young mycorrhizal pedunculate oak trees (Quercus robur L.)

Uptake and xylem loading of organic sulfur and nitrogen were analyzed in detached mycorrhizal (Laccaria laccata L.) roots of pedunculate oak (Quercus robur L.) seedlings using radiolabeled reduced glutathione (GSH) and glutamine (Gln) for transport analyses. The experiments showed for the first time that GSH is taken up by plant roots from the nutrient solution and is partially allocated to the shoot. Apparently, GSH produced during mineralization processes in the soil can be used by plant roots as a sulfur source. GSH uptake into the roots showed biphasic kinetics within the concentration range studied (0–500 M) with maximum transport velocities (v _max) and substrate affinities (K _m) that were similar to the kinetics of Gln uptake. GSH uptake kinetics were also in the same range as previously reported for sulfate uptake by mycorrhizal roots of pedunculate oak. It may therefore be assumed that GSH and sulfate uptake can be of comparable significance for sulfur nutrition, provided both sulfur sources are available at similar concentrations at the sites of uptake. Xylem loading of GSH and Gln showed monophasic transport kinetics with v _max significantly lower than observed for the two respective uptake systems and, as indicated by the K _m-values, a substrate affinity between the high and the low affinity uptake systems. The possible nature of the transport systems for GSH and Gln is discussed.     

Interactive effects of mycorrhization and elevated carbon dioxide on growth of young pedunculate oak (Quercus robur L.) trees

Pedunculate oak (Quercus robur L.) was germinated and grown at ambient CO₂ level and 650 ppmv CO₂ in the presence and absence of the ectomycorrhizal fungus Laccaria laccata for a total of 6 month under nutrient non-limiting conditions. Mycorrhization and elevated atmospheric CO₂ each supported the growth of the trees. Stem height, stem diameter, and dry matter accumulation of pedunculate oak were increased by mycorrhization. Elevated atmospheric CO₂ enhanced stem height, stem diameter, fresh weight and dry weight, as well as lateral root formation of the trees. In combination, mycorrhization and elevated atmospheric CO₂ had a more than additive, positive effect on tree height and biomass accumulation, and further improved lateral root formation of the trees. From these findings it is suggested that the efficiency of the roots in supporting the growth of the shoot is increased in mycorrhized oak trees at elevated atmospheric CO₂.Abbreviations DW dry weight - FW fresh weight - RWC relative water content     

Epigenetic characterization of chromatin in cycling cells of pedunculate oak, Quercus robur L.

Cycling cells of Quercus robur have a simple nuclear organization where most of the heterochromatin is visible as DAPI-positive chromocenters, which correspond to DAPI bands at the (peri)centromeric region of each of the 24 chromosomes of the oak complement. Immunofluorescence using 5-mC revealed dispersed distribution of the signal throughout the interphase nucleus/chromosomes without enrichment within DAPI-positive chromocenters/bands, suggesting that DNA methylation was not restricted to constitutive heterochromatin, but was associated with both euchromatic and heterochromatic domains. While H3K9ac exhibited typical euchromatin-specific distribution, the distributional pattern of histone methylation marks H3K9me1, H3K27me2, and H3K4me3 showed some specificity. The H3K9me1 and H3K27me2, both heterochromatin-associated marks, were not restricted to chromocenters, but showed additional dispersed distribution within euchromatin, while H3K27me2 mark also clustered in foci that did not co-localize with chromocenters. Surprisingly, even though H3K4me3 was distributed in the entire chromatin, many chromocenters were enriched with this euchromatin-specific modification. We discuss the distribution of the epigenetic marks in the context of the genome composition and lifestyle of Q. robur.     

Synchronization of larval emergence in winter moth (Operophtera brumata L.) and budburst in pedunculate oak (Quercus robur L.) under simulated climate change

1. The hypothesis that a 3 °C elevation in temperature and doubled CO₂ concentration would have no effect on the synchronization of winter moth egg hatch with budburst in oak was tested by comparing the separate and interactive effects of ambient and elevated (+ 3 °C) temperature and ambient and elevated (doubled to 340 p.p.m.) CO₂ in eight experimental Solardomes. In addition, an outdoor control was compared with the ambient temperature/CO₂ treatment combination.
2. Elevated temperature accelerated darkening (preceding egg hatch by about 5–10 days) and hatching of eggs developing off the trees; elevated CO₂ had no effect. The same effects were observed in eggs developing on the trees.
3. Within treatments, date of egg hatch was the same on trees with early or late budburst.
4. Egg darkening and budburst were closely synchronized at both ambient and elevated temperatures.
5. Both eggs and trees required fewer cumulative heat units (day degrees > 4 °C), for hatching and budburst, respectively, at ambient than elevated temperatures. The requirements in the outdoor control treatment were similar to those in the ambient Solardome treatment.
6. Egg hatch between 10 and 25 °C, on a temperature gradient in the laboratory, required a constant number of heat units; fewer were required below 10 °C.
7. Elevated temperatures, in the Solardomes and the field, delayed adult emergence from the pupae.
8. The results suggest that a general increase in temperature with climatic change would not affect the closeness of the synchronization between egg hatch of winter moth and budburst of oak.     

Genetic variability of fragmented stands of pedunculate oak (Quercus robur) in Finland

The genetic structure of 33 natural Quercus robur stands in Finland was studied using 13 allozyme loci to analyze the effects of fragmentation in a wind-pollinated tree species. The present fragmented and discontinuous distribution of oak is a result of both short-term human impact and long-term climatic and geological change, including post-glacial land uplift. In accordance with general expectations, genetic diversity in small populations was lower than that in large populations, and differentiation among small populations was higher than that among large populations. Heterozygote deficiency was more pronounced in large populations, which is proposed to be a Wahlund effect created by either spatial sub-structuring or the existence of synchronized flowering lineages. Also genetic differentiation was higher and diversity lower in Finland than the estimates reported for Central Europe. There were differences in the genetic structure on sites of different geological age. We suggest that on most geologically old sites drift has a prominent effect whereas on younger sites also founder effects may be important.     

Temporal dynamics of allelic diversity in isolated population of pedunculate oak Quercus robur L. (Fagaceae)

Using nine microsatellite loci, genetic diversity of small geographically isolated population of pedunculate oak Quercus robur L. (Fragaceae) was examined. The population was located outside of the species range in Bashkir Transuralia. Considerable temporal dynamics of allelic diversity, explained in terms of different effectiveness of gene flow in different years, was demonstrated.     

Fine spatial structure of allozyme genotypes in isolated population of pedunculate oak Quercus robur L. (Fagaceae)

The extent and spatial pattern of genetic variation at polymorphic allozyme loci in a population of pedunculate oak Quercus robur from the Bashkir Transural region was investigated using autocorrelation analysis. In the plantation examined, statistically significant local concentration of most of the alleles in two-dimensional space was identified. The measures for protection of this small population located outside of the western border of the species range, in the mountain--steppe habitat, and characterized by specific gene pool, are suggested.     

Detection of microsatellite instability during somatic embryogenesis of oak (Quercus robur L.)

Five microsatellite loci (QpZAG1/5, QpZAG9, QpZAG36, MSQ4, MSQ13) were used to test for genetic stability of three somatic embryogenic culture lines of Quercus robur L. and plantlets derived therefrom. DNA variation was detected among somatic embryos within all embryogenic lines, whereas no genetic instability was found among the regenerated plants. Two microsatellite loci revealed variation, and a locus-dependent instability was observed. The most polymorphic and useful microsatellite locus for detecting genetic variation was QpZAG9, with 28.5% of the investigated loci being variable.     

Growth and nutation parameters of primary root of pedunculated oak (Quercus robur L.)

The elongation of primary roots of pedunculated oak (Quercus robur L.), which is associated with nutation oscillations, was registered by a time lapse cinematography. The following characteristics were expressed graphically: relative growth rate [mm h-¹], frequency [rev. h-¹] and amplitudes [mm] of nutation oscillations, their direction and location of nutation curvatures. The growth-oscillation mechanism is interpreted as a part of the geocontrol system of the root and was evaluated on the basis of its physiological efficiency. Comparison of nutation periods showed that the control system of pedunculated oak is three times more and two times less efficient than that of pea and maize, respectively. Comparison of the oscillation amplitudes of growing roots gives similar results.     

Severe winter rings of oak trees (Quercus robur L.) from Central European Russia

Oak trees were sampled in a flood plain forest in the valley of the Zapadnaya Dvina (Daugava) river (Tver region, Russia). Annual rings of the time period from 1826 to 2010 were studied. Anatomically distinct rings with a stripe of small-sized cells in the innermost part and narrow earlywood vessels located in three to four rows occurred in 1861, 1862, 1929, 1940, 1942, 1956 and 1979. Deviations of earlywood development were associated with the drop of winter temperature below ?42 °C. The percentage of severe winter ring (SWR) occurrence depends upon tree age and decreases from 75.6 % in younger specimens (under 41 years old at the time of the severe winter) to 27.1 % in middle-aged ones (from 41 to 80 years) to 3.5 % in trees older than 80 years. Described anatomical features can be used in the reconstruction of severe winter frequency in the past.     

The content of abscisic acid in the developing seeds of oak (Quercus robur L.)

Abscisic acid was analysed in seeds of oak, at. five stages of development in threeweek intervals, starting on the 6th week after anthesis until maturation in the 18th week after anthesis. The physico-chemical methods and bioassays were used for a closer characterization and quantification of the examined compounds. Differentiation of the ABA level depending upon the stages of seed development has been observed in various parts of acorn. Presented at the International Symposium “Plant Growth Regulators” held on June 18 – 22, 1984 at Liblice, Czechoslovakia.     

Genetic differentiation of pedunculate oak Quercus robur L. in the European part of Russia based on RAPD markers

Using randomly amplified polymorphic DNA markers (RAPD), genetic variation and differentiation in four populations of pedunculate oak Quercus robur L. were examined. The populations occupy a large part of the Quercus robur range in the European Russia (Voronezh and Novgorod oblasts; Republics of Mordovia and Bashkortostan). With each of six random primers (A02, A09, A17, B01, B08, B11), 96 DNA samples were analyzed by PCR. In all, 48 putative polymorphic RAPD loci were detected. We failed to reveal population-specific DNA fragments for any primer although the frequencies of 14 fragments were significantly different among populations. The oak populations studied exhibited high variability: 73-90% of genes were polymorphic and the effective allele number was about 1.4. The total genetic variation varied from 0.202 (Vor) to 0.245 (Nov), which corresponded to the estimates for populations of this species from Central and Western Europe. The populations examined showed low among-population differentiation (GST = 0.098); gene flow Nem was 4.61. The proportion of among-population variation of the RAPD loci studied accounted for 7% of the total variability; more than 93% of the total variability was explained by individual and within--population variation.     

The alpha-tocopherol content of leaves of pedunculate oak (Quercus robur L.)--variation over the growing season and along the vertical light gradient in the canopy

This study was performed in order to investigate whether the actual requirement for defence against photo-oxidative stress is reflected by the alpha-tocopherol (alpha-Toco) content in leaves of pedunculate oak (Quercus robur L.). Antioxidants and pigments were quantified in leaves that were collected on six days between May and September 2000 in a mixed pine/oak forest at canopy positions differing in light environment. Pools of hydrophilic antioxidants and photo-protective xanthophyll cycle pigments (V + A + Z) reflected the anti-oxidative demand, as these pools increased with the average light intensity to which the leaves were acclimated. The photo-protective demand was not the determinant of the alpha-Toco content of oak leaves, as (1) foliage of a young oak, exposed to low light levels in the understorey, contained higher amounts of this lipophilic antioxidant than leaves sampled from semimature oaks at canopy positions with a similar light environment, and (2) a strong increase in the alpha-Toco content over the growing season was detected at each investigated crown position, whereas the V + A + Z pool did not show a concomitant accumulation during leaf ageing. The rate of alpha-Toco accumulation differed distinctly between samples taken at different canopy positions.     

Interactive effects of mycorrhization and elevated atmospheric CO2 on sulphur nutrition of young pedunculate oak (Quercus robur L.) trees

Pedunculate oak (Quercus robur L.) was germinated and grown at ambient CO₂ concentration and 650 μmol mol^?1 CO₂ in the presence and absence of the ectomycorrhizal fungus Laccaria laccata for a total of 22 weeks under nonlimiting nutrient conditions. Sulphate uptake, xylem loading and exudation were analysed in excised roots. Despite a relatively high affinity for sulphate (K_M= 1.6 mmol m^?3), the rates of sulphate uptake by excised lateral roots of mycorrhizal oak trees were low as compared to herbaceous plants. Rates of sulphate uptake were similar in mycorrhizal and non-mycorrhizal roots and were not affected by growth of the trees at elevated CO₂. However, the total uptake of sulphate per plant was enhanced by elevated CO₂ and further enhanced by elevated CO₂ and mycorrhization. Sulphate uptake seemed to be closely correlated with biomass accumulation under the conditions applied. The percentage of the sulphate taken up by mycorrhizal oak roots that was loaded into the xylem was an order of magnitude lower than previously observed for herbaceous plants. The rate of xylem loading was enhanced by mycorrhization and, in roots of mycorrhizal trees only, by growth at elevated CO₂. On a whole-plant basis this increase in xylem loading could only partially be explained by the increased growth of the trees. Elevated CO₂ and mycorrhization appeared to increase greatly the sulphate supply of the shoot at the level of xylem loading. For all treatments, calculated rates of sulphate exudation were significantly lower than the corresponding rates of xylem loading of sulphate. Radiolabelled sulphate loaded into the xylem therefore seems to be readily diluted by unlabelled sulphate during xylem transport. Allocation of reduced sulphur from oak leaves was studied by flap-feeding radiolabelled GSH to mature oak leaves. The rate of export of radioactivity from the fed leaves was 4–5 times higher in mycorrhizal oak trees grown at elevated CO₂ than in those grown at ambient CO₂. Export of radiolabel proceeded almost exclusively in a basipetal direction to the roots. From these experiments it can be concluded that, in mycorrhizal oak trees grown at elevated CO₂, the transport of sulphate to the shoot is increased at the level of xylem loading to enable increased sulphate reduction in the leaves. Increased sulphate reduction seems to be required for the enhanced allocation of reduced sulphur to the roots which is observed in trees grown at elevated CO₂. These changes in sulphate and reduced sulphur allocation may be a prerequisite for the positive effect of elevated CO₂ on growth of oak trees previously observed.