Sporoderm characters ofTetrameranthus duckei (Annonaceae) and their systematic implications

Ultrastructural and micromorphological investigations of the sporoderm ofTetrameranthus duckei pollen confirm the interpretation of this genus as being isolated within theAnnonaceae.     

Comparing EST-based genetic maps between <Emphasis Type="BoldItalic">Pinus sylvestris</Emphasis> and <Emphasis Type="BoldItalic">Pinus taeda</Emphasis>

A genetic map of Pinus sylvestris was constructed using ESTP (expressed sequence tag polymorphism) markers and other gene-based markers, AFLP markers and microsatellites. Part of the ESTP markers (40) were developed and mapped earlier in Pinus taeda, and additional markers were generated based on P. sylvestris sequences or sequences from other pine species. The mapping in P. sylvestris was based on 94 F₁ progeny from a cross between plus-tree parents E635C and E1101. AFLP framework maps for the parent trees were first constructed. The ESTP and other gene sequence-based markers were added to the framework maps, as well as five published microsatellite loci. The separate maps were then integrated with the aid of AFLPs segregating in both trees (dominant segregation ratios 3:1) as well as gene markers and microsatellites segregating in both parent trees (segregation ratios 1:1:1:1 or 1:2:1). The integrated map consisted of 12 groups corresponding to the P. taeda linkage groups, and additionally three and six smaller groups for E1101 and E635C, respectively. The number of framework AFLP markers in the integrated map is altogether 194 and the number of gene markers 61. The total length of the integrated map was 1,314 cM. The set of markers developed for P. sylvestris was also added to existing maps of two P. taeda pedigrees. Starting with a mapped marker from one pedigree in the source species resulted in a mapped marker in a pedigree of the other species in more than 40% of the cases, with about equal success in both directions. The maps of the two species are largely colinear, even if the species have diverged more than 70 MYA. Most cases of different locations were probably due to problems in identifying the orthologous members of gene families. These data provide a first ESTP-containing map of P. sylvestris, which can also be used for comparing this species to additional species mapped with the same markers.Communicated by C. Möllers     

Brown rot decay of copper-chromated-phosphorus impregnated fence poles: Characterization by molecular analyses and microscopy

At one location in central Sweden, agricultural pine (Pinus sylvestris L.) fence poles treated with a commercial copper–chromium–phosphorus preservative (CCP) formulation according to use class 4 at retention of 30 kg m⁻³ were prematurely degraded by fungi after only two years in-service. Light- and electron microscopy analyses showed decay to result from primarily brown rot attack. Culture studies produced on different agar and copper-containing media using small wood slivers removed from infected poles allowed establishment of a number of pure cultures of Phycomycetes, Ascomycetes, Fungi Imperfecti and Basidiomycete fungi. Using morphological characters, PCR and sequencing of isolated strains, Antrodia vaillantii was determined as the most abundant basidiomycete present and as the major causal agent of decay. Compatibility tests and comparison of the ITS nrDNA sequences of our putative A. vaillantii isolate with other A. vaillantii strains and with Antrodia radiculosa showed differences suggesting a hybrid strain. A combination of site characteristics (e.g. hot spots of A. vaillantii), the use of juvenile poles, copper tolerance and overall ineffectivity of CCP against A. vaillantii is suggested as reasons for premature decay.     

New insights from MALDI-ToF MS, NMR, and GC-MS: mass spectrometry techniques applied to palynology

Summary. The present study for the first time describes the application of matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-ToF MS) to palynology. With an accessible mass range of up to about 350,000 Da at subpicomolar range, this technique is ideal for the characterisation of bio-macromolecules, such as sporopollenin, found in fossil and extant pollen and spore walls, which often can only be isolated in very small quantities. At this stage, the limited solubility of sporopollenin allows for the identification of sections of this biopolymer, but with the optimisation of MALDI-ToF matrices, further structure elucidation will become possible. Furthermore, gas chromatography–mass spectrometry (GC-MS) and ¹H nuclear magnetic resonance (¹H NMR) spectroscopy data obtained from a number of experiments revealed that some previously reported data were misinterpreted. These results add support to the hypothesis that common plasticizers were wrongly described as sporopollenin compounds. Correspondence and reprints: School of Earth, Ocean and Planetary Sciences, Cardiff University, Park Place, Cardiff CF10 3YE, United Kingdom. Permanent address: Albion College, Albion, Michigan, U.S.A.     

Effects of drought and waterlogging on ultrastructure of Scots pine and Norway spruce needles

Effects of water stress on needle ultrastructure of 2-year-old Scots pine (Pinus sylvestris L.) and 5-year-old Norway spruce [Picea abies (L.) Karst.] seedlings were studied in greenhouse experiments. Drought stress was induced by leaving seedlings without watering, and waterlogging stress was produced by submerging the seedling containers in water. Needle samples for ultrastructural analyses were collected several times during the experiments, and samples for nutrient analyses at the end of the experiments. In drought stress, plasmolysis of mesophyll and transfusion parenchyma tissues, aggregation of chloroplast stroma and its separation from thylakoids and decreased size and abundance of starch grains in needles of both species were observed. The concentration of lipid bodies around the chloroplasts were detected in pine needles. Calcium and water concentrations in spruce needles were lower by the end of the experiments compared to controls. In waterlogging treatment, swelling of phloem cells in pine needles and large starch grains, slight swelling of thylakoids and increased translucency of plastoglobuli in chloroplasts of both species studied were observed. The phosphorus concentration in pine needles was higher while phosphorus, calcium and magnesium concentrations in spruce needles were lower in the waterlogging treatments compared to controls. Typical symptoms induced by drought stress, e. g. aggregation of chloroplast stroma and its separation from thylakoids, were detected, but, in waterlogging stress, ultrastructural symptoms appeared to be related to the developing nutrient imbalance of needles.     

Spermidine and the ectomycorrhizal fungus Pisolithus tinctorius synergistically induce maturation of Scots pine embryogenic cultures

Exogenous spermidine (Spd) and the ectomycorrhizal (ECM) fungus Pisolithus tinctorius (Pers.) Coker and Couch had a synergistic effect on the maturation of Scots pine (Pinus sylvestris L.) somatic embryos. Induced maturation was expressed as a higher number of cell masses able to form embryos and a greater number of embryos formed per cell mass. In contrast, treatment with P. tinctorius alone on the hormone-free medium resulted in the lowest embryo-forming capacity. Retarded proliferation growth appeared to be required for maturation, but did not explain the synergistic effect of the fungus and exogenous Spd. Simultaneous treatment did not result in lower concentrations of putrescine (Put), Spd or spermine (Spm) in the embryogenic cell masses relative to the separate treatments. Our study is the first report on the use of a specific ECM fungus to induce maturation of somatic embryos, and it indicates that P. tinctorius was able to modify the maturation media in a way that, together with exogenous Spd, positively affected embryogenic cultures of Scots pine. Our study also shows that it is possible to enhance plant development other than root formation by using specific ECM fungi.     

Regulation of photoassimilate allocation in Pinus sylvestris seedlings by the nutritional status of the mycorrhizal fungus Suillus variegatus

Summary Seedlings of Pinus sylvestris L. were grown on defined nutrient solutions on carbon filters, either sterile or infected with the basidiomycete Suillus variegatus O. Kuntze. After mycorrhizas were established, the shoot of the seedling was subjected to ¹⁴CO₂ photosynthesis. ¹⁴C-labelled photoassimilates were translocated to both mycorrhizas and non-infected root tips. Microautoradiographs of mycorrhizas indicated that omission of external sugars did not affect the formation of mycorrhizas; ¹⁴C-photoassimilates were supplied to cortex, Hartig net and the mantle of hyphae surrounding the rootlet. Nutrient solution containing sugars (malt extract, glucose) enhanced the growth of the fungus. As a consequence, ¹⁴C-photoassimilates from the seedling were accumulated in the mantle, but defence mechanisms of the host cannot be excluded. When soluble nitrogen was omitted from the nutrient solution and replaced by chitin precipitated on the filter-bearing mycorrhizas, the fungus appeared strongly labelled in the mantle, where the fungal chitinase provided soluble nitrogen compounds, necessary for the growth of the seedling.     

Spermidine and methylglyoxal bis(guanylhydrazone) affect maturation and endogenous polyamine content of Scots pine embryogenic cultures

Exogenous spermidine (Spd) and methylglyoxal bis(guanylhydrazone) (MGBG), a putative inhibitor of Spd synthesis, improved somatic embryo formation of Scots pine (Pinus sylvestris L.). The induced maturation due to MGBG and Spd was accompanied by significantly retarded proliferation growth and by reduction in the concentration of free polyamines compared to the control cultures. The action of MGBG revealed that it has a non-specific effect on the whole polyamine metabolism of Scots pine. Furthermore, at certain concentrations it may induce plant differentiation as well.     

The ancient use ofPinus sylvestris L. (Scots pine) inner bark by Sami people in northern Sweden, related to cultural and ecological factors

A multidisciplinary study of use by Sami people ofPinus sylvestris L. (Scots pine) inner bark was performed in northern Sweden. We combined linguistic, historical and archaeological records with ecological data collected from field studies to investigate important cultural and ecological factors associated with previous use of bark. Our data from bark peeled trees at 313 sites were used to describe patterns in space and time in the land-scape. Sites with bark peeled trees were mainly found in the central and northern part of Lapland (65°00–68°14N). Large numbers of barked trees were found in undisturbed forests in national parks and reserves but few were found in forests under commercial management. The dendroecological analysis revealed a continuous use of inner bark from A.D. 1450 to 1890. Large sheets of bark were taken from trees in the spring, prepared and stored as a staple food resource. Inner bark was eaten fresh, dried or roasted. Smaller bark peelings were used for the wrapping of sinews. The cessation of bark use in the 19th century was driven by several factors, but the availability of other products that could replace traditional use of bark was specifically important. The previous common and varied use of bark, the great age of Sami terminology and a possible association with archaeological remains of potential great age indicate that peeled bark was used long before the historic period. Our study also focused on the ongoing loss of culturally-modified trees in forests outside protected areas.     

An ultrastructural,cytochemical and immunofluorescence study of postmeiotic development of plasmodial tapetum inTradescantia virginiana L. and its relevance to the pathway of sporopollenin secretion

Summary Establishment of a tapetal plasmodium in postmeiotic stages in anther locules ofTradescantia virginiana encloses the tetrads in membrane-limited compartments. The perispore membrane (PSM), around each tetrad, is derived from composite tapetal cell plasma membranes. The tapetum acquires an abundance of ER and ribosomes and by the late tetrad stage the PSM and its underlying cytoplasm exhibit specialized features, studied here by ZnIO impregnation, osmium maceration, application of indirect immunofluorescence employing antitubulin, conventional thin sectioning and the Thiéry reaction. These features include: labyrinthine convolutions of the PSM resulting from migration of membranous sacs and their partial fusion to the PSM, an intimate relationship of tubular ER with the convoluted PSM, and microtubules underlying the PSM and among the membranous sacs. At the same time membrane-bound granules, comparable to but smaller and simpler than tapetal orbicules of secretory tapeta, form in the convolutions. It is postulated that the ER supplies precursors of sporopollenincontaining parts of the spore wall, that the PSM-associated microtubules stabilise the whole secretory apparatus at the tapetum-spore interface, and that the precursors are expelled into the lumen bounded by the PSM and then accreted upon the orbicule-like granules or the developing spore wall. With dissolution of the callosic wall, the plasmodium invades the intermicrosporal spaces of late tetrads, the PSM unfolding its elaborations and becoming closely appressed to the exinous surfaces of individual spores. Microtubules, although present during this phase of invasion, do not seem to propel the invasion processes and may have roles in shape maintenance. During pollen mitosis and enlargement the tapetal cytoplasm accumulates lipidic globules. A late phase of Golgi activity precedes accumulation of vesicles or vacuoles near the spores, these being bounded by single or multiple tripartite membranes. With anther desiccation, portions of plasmodium are deposited on the pollen surface in the form of tryphine, the deposits containing stacked membrane-like bilayers.     

Response of understorey vegetation and Scots pine root systems to fertilization at multiple deficiency stress

The stump and root systems of Scots pine (Pinus sylvestris L.) and field-layer vegetation were sampled before and three growing seasons after drainage and fertilization of a low-shrub pine bog in SE Norway. Although the understorey vegetation roots responded significantly to nutrient application with higher concentrations of Ca and P, root biomass weights did not change. The fine and small pine roots responded with higher N, Ca, P and S concentrations, while those of Mn and Zn were significantly reduced. The NPK-application resulted in significantly higher pine root biomass. Relative to the total stores in the root zone the amounts of most elements in roots shifted to higher ratios at NPK-application. High figures for K, B and Mn indicate tight biochemical cycles of these elements. Compared to totals in above and below ground biomass, major parts of Fe and Pb were held by the roots. In contrast the field layer roots kept a very small per cent of total K, while the pine roots were low in Mn. The understorey vegetation was primarily restricted by P-deficiency, while the pine trees were also restricted by low supply of N. The field and the tree layer species seem to differ with respect to required nutrient concentrations in the root zone. These characteristics are important for direction and extent of successional changes after fertilization in low-shrub pine bog ecosystems.     

Pollen morphology of the cypripedioid orchids

Pollen morphology of the four traditional genera (Paphiopedilum, Selenipedium, Cypripedium, Phragmipedium) comprising the cypripedioid orchids indicates that the monads are sulcate, more or less smooth-surfaced, and covered by a non-acetolysis resistant layer called elastoviscin. Evidence from ultrathin sections of pollen grains shows that typical exine layers are present only inSelenipedium, modified inPhragmipedium and absent inPaphiopedilum and most species ofCypripedium; that a small, inconspicuous portion of the grain surface is constructed as a sulcus; and that the pollen grain wall acts as a sponge in rapidly absorbing water. Several instances of parallelisms between non-related families and among different groups of orchids are reported and new ideas on the evolution of theCypripedioideae are presented.Dedicated to Prof. DrE. Tschermak-Woess on the occasion of her 70th birthday.Reprint requests toM. Hesse.     

Triphenyltetrazolium chloride as an indicator of fine-root vitality and environmental stress in coniferous forest stands: Applications and limitations

The present study is an attempt to investigate whether triphenyltetrazolium chloride (TTC), a chemical compound which measures dehydrogenase activity, could be used to study fine-root vitality from two different points of view: (i) in relation to ageing; (ii) as an indicator of environmental stress, in this case of excess nitrogen. The study was performed with excavated fine-roots from middle-aged Norway spruce and Scots pine stands.The ageing aspect was investigated by applying TTC to fine roots separated into different vitality classes, based on certain morphological characteristics. A significant difference in activity was demonstrated only in the case of roots that could be referred to as living and dead, respectively. The use of TTC on fine roots grown at different nitrogen supply levels indicates a possible increase in dehydrogenase activity with increasing nitrogen supply.     

Lignosulfonate promotes the interaction between Scots pine and an ectomycorrhizal fungus Pisolithus tinctorius in vitro

Lignosulfonate (LS) is a lignin-based polymer obtained as a by-product from paper industry, which may have potential as an amendment with macronutrients. We studied effects of LS on the interaction between Scots pine (Pinus sylvestris L.) seedlings and hypocotyl cuttings and the ectomycorrhizal (ECM) fungusPisolithus tinctorius (Pers.) Coker and Couch. The experiments were performed in vitroon the MMN agar medium containing Fe–LS chelate at the concentrations of 0, 5, 10 and 25 mg/L. Inoculation with P. tinctoriusincreased root growth of the seedlings. Fe–LS enhanced P. tinctorius induced formation of lateral roots and had a dose-dependent positive effect on the establishment of mycorrhizas on the seedlings. The growth of the fungal mycelium was improved by Fe–LS, which might cause faster and more intensive contact with the roots and, thus, better root growth and mycorrhiza formation. P.tinctorius enhanced also adventitious root formation and subsequent root growth of the hypocotyl cuttings but without any synergistic effect with Fe–LS. Our study with P. tinctorius and Scots pine in vitro indicates that a low-cost by-product Fe–LS, obtained from paper industry, may be a potential tool to improve the efficiency of fungal inoculations, thus, facilitating the early interaction between an ECM fungus and host seedling.