Pyrus pyrifolia stylar S-RNase induces alterations in the actin cytoskeleton in self-pollen and tubes in vitro

Summary. Pears (Pyrus pyrifolia L.) have an S-RNase-based gametophytic self-incompatibility system, and S-RNases have also been implicated in self-pollen or genetically identical pollen rejection. Tip growth of the pollen tube is dependent on a functioning actin cytoskeleton. In this study, configurations of the actin cytoskeleton in P. pyrifolia pollen and effects of stylar S-RNases on its dynamics were investigated by fluorescence and confocal microscopy. Results show that actin filaments in normal pollen grains exist in fusiform or circular structures. When the pollen germinates, actin filaments assembled around one of the germination pores, and then actin bundles oriented axially throughout the shank of the growing tube. There was a lack of actin filaments 5–15 μm from the tube tip. When self-stylar S-RNase was added to the basal medium, pollen germination and tube growth were inhibited. The configuration of the actin cytoskeleton changed throughout the culturing time: during the first 20 min, the actin configurations in the self-pollen and tube were similar to the control; after 20 min of treatment, the actin filaments in the pollen tube gradually moved into a network running from the shank to the tip; finally, there was punctate actin present throughout the whole tube. Although the actin filaments of the self-pollen grain also disintegrated into punctate foci, the change was slower than in the tube. Furthermore, the alterations to the actin cytoskeleton occurred prior to the arrest of pollen tube growth. These results suggest that P. pyrifolia stylar S-RNase induces alterations in the actin cytoskeleton in self-pollen grains and tubes. Correspondence: Shao-ling Zhang, College of Horticulture, Nanjing Agricultural University, Nanjing, Jiangsu 210095, People’s Republic of China.     

The actin cytoskeleton in unfixed pollen tubes following microwave-accelerated DMSO-permeabilisation and TRITC-phalloidin staining

Summary The disposition of the actin cytoskeleton in pollen tubes of Narcissus pseudonarcissus has been investigated using microwave-accelerated DMSO-permeabilisation and TRITC-phalloidin (Tr-Ph) staining. Extending tubes were transferred from growth medium into a calcium-free medium containing 1 g ml^–1 Tr-Ph and 5% DMSO. After 10 s irradiation in a 500 W microwave oven, when the temperature in the sample was estimated at 52° C, some two-thirds of the tubes retained essentially normal apical zonation; in the remainder the cytoplasm was coarsely granular, and the zonation had been lost. Optimal Tr-Ph staining of the actin cytoskeleton was obtained about 1 h after irradiation. In the most favourable cases, the transition from a longitudinally oriented system of fine fibrils in the sub-apical region to a mass of shorter fibrils in the centre of the apex could be traced, with a peripheral population of more extended fibrils continuing further along the forming wall towards the growing point. This organisation can be reconciled with that revealed in recent fine-structural studies of the microfilament system of the pollen tube apex using freeze-substitution. The relationship of the system with the pattern of movement observed in the apical region of the living tube and with the probable mechanism of tip growth is briefly discussed.     

The actin microfilament network within elongating pollen tubes of the gymnospermPicea abies (Norway spruce)

Summary Actin microfilaments form a dense network within pollen tubes of the gymnosperm Norway spruce (Picea abies). Microfilaments emanate from within the pollen grain and form long, branching arrays passing through the aperture and down the length of the pollen tube to the tip. Pollen tubes are densely packed with large amyloplasts, which are surrounded by branching microfilament bundles. The vegetative nucleus is suspended within the elongating pollen tube within a complex array of microfilaments oriented both parallel to and perpendicular with the growing axis. Microfilament bundles branch out along the nuclear surface, and some filaments terminate on or emanate from the surface. Microfilaments in the pollen tube tip form a 6 m thick, dense, uniform layer beneath the plasma membrane. This layer ensheathes an actin depleted core which contains cytoplasm and organelles, including small amyloplasts, and extends back 36 m from the tip. Behind the core region, the distinct actin layer is absent as microfilaments are present throughout the pollen tube. Organelle zonation is not always maintained in these conifer pollen tubes. Large amyloplasts will fill the pollen tube up to the growing tip, while the distinct layer of microfilaments and cytoplasm beneath the plasma membrane is maintained. The distinctive microfilament arrangement in the pollen tube tips of this conifer is similar to that seen in tip growth in fungi, ferns and mosses, but has not been reported previously in seed plants.     

Cytochalasin B Treatment of Apple (<Emphasis Type="Italic">Malus pumila</Emphasis> Mill.) Pollen Tubes Alters the Cytoplasmic Calcium Gradient and Causes Major Changes in the Cell Wall Components

It is well established that the actin cytoskeleton is absolutely essential to pollen germination and tube growth. In this study we investigated the effects of cytochalasin B (CB), which affects actin polymerization by binding to the barbed end of actin filaments, on apple (Malus pumila Mill.) pollen tube growth. Results showed that CB altered the morphology of pollen tubes, which had a larger diameter than control tubes beside inhibiting pollen germination and tube growth. Meantime CB also caused an abnormal distribution of actin filaments in the shank of the treated pollen tubes. Fluo-3/AM labeling indicated that the gradient of cytosolic calcium ([Ca²⁺]c) in the pollen tube tip was abolished by exposure to CB, which induced a much stronger signal in the cytoplasm. Cellulose and callose distribution in the tube apex changed due to the CB treatment. Immunolabeling with different pectin and arabinogalactan protein (AGP) antibodies illustrated that CB induced an accumulation of pectins and AGPs in the tube cytoplasm and apex wall. The above results were further supported by Fourier-transform infrared (FTIR) analysis. The results suggest the disruption of actin can result in abnormal growth by disturbing the [Ca²⁺]c gradient and the distribution of cell wall components at the pollen tube apex.     

Imaging the actin cytoskeleton in growing pollen tubes

Given the importance of the actin cytoskeleton to pollen tube growth, we have attempted to decipher its structure, organization and dynamic changes in living, growing pollen tubes of Nicotiana tabacum and Lilium formosanum, using three different GFP-labeled actin-binding domains. Because the intricate structure of the actin cytoskeleton in rapidly frozen pollen tubes was recently resolved, we now have a clear standard against which to compare the quality of labeling produced by these GFP-labeled probes. While GFP-talin, GFP-ADF and GFP-fimbrin show various aspects of the actin cytoskeleton structure, each marker produces a characteristic pattern of labeling, and none reveals the entire spectrum of actin. Whereas GFP-ADF, and to a lesser extent GFP-talin, label the fringe of actin in the apex, no similar structure is observed with GFP-fimbrin. Further, GFP-ADF only occasionally labels actin cables in the shank of the pollen tube, whereas GFP-fimbrin labels an abundance of fine filaments in this region, and GFP-talin bundles actin into a central cable in the core of the pollen tube surrounded by a few finer elements. High levels of expression of GFP-talin and GFP-fimbrin frequently cause structural rearrangements of the actin cytoskeleton of pollen tubes, and inhibit tip growth in a dose dependent manner. Most notably, GFP-talin results in thick cortical hoops of actin, transverse to the axis of growth, and GFP-fimbrin causes actin filaments to aggregate. Aberrations are seldom seen in pollen tubes expressing GFP-ADF. Although these markers are valuable tools to study the structure of the actin cytoskeleton of growing pollen tubes, given their ability to cause aberrations and to block pollen tube growth, we urge caution in their use. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. Financial Source: National Science Foundation grant Nos. MCB-0077599 and MCB-0516852 to PKH EU Research Training Network TIPNET (project HPRN-CT-2002-00265), Brussels, Belgium, to BV     

Centrifugation causes adaptation of microfilaments Studies on the transport of statoliths in gravity sensingChara rhizoids

Summary The actin cytoskeleton is involved in the positioning of statoliths in tip growingChara rhizoids. The balance between the acropetally acting gravity force and the basipetally acting net out-come of cytoskeletal force results in the dynamically stable position of the statoliths 10–30 m above the cell tip. A change of the direction and/or the amount of one of these forces in a vertically growing rhizoid results in a dislocation of statoliths. Centrifugation was used as a tool to study the characteristics of the interaction between statoliths and microfilaments (MFs). Acropetal and basipetal accelerations up to 6.5 g were applied with the newly constructed slow-rotating-centrifuge-microscope (NIZEMI). Higher accelerations were applied by means of a conventional centrifuge, namely acropetally 10–200 g and basipetally 10–70 g. During acropetal accelerations (1.4–6 g), statoliths were displaced to a new stable position nearer to the cell vertex (12–6.5 m distance to the apical cell wall, respectively), but they did not sediment on the apical cell wall. The original position of the statoliths was reestablished within 30 s after centrifugation. Sedimentation of statoliths and reduction of the growth rates of the rhizoids were observed during acropetal accelerations higher than 50 g. When not only the amount but also the direction of the acceleration were changed in comparison to the natural condition, i.e., during basipetal accelerations (1.0–6.5 g), statoliths were displaced into the subapical zone (up to 90 m distance to the apical cell wall); after 15–20 min the retransport of statoliths to the apex against the direction of acceleration started. Finally, the natural position in the tip was reestablished against the direction of continuous centrifugation. Retransport was observed during accelerations up to 70 g. Under the 1 g condition that followed the retransported statoliths showed an up to 5-fold increase in sedimentation time onto the lateral cell wall when placed horizontally. During basipetal centrifugations 70 g all statoliths entered the basal vacuolar part of the rhizoid where they were cotransported in the streaming cytoplasm. It is concluded that the MF system is able to adapt to higher mass accelerations and that the MF system of the polarly growing rhizoid is polarly organized.Abbreviations g gravitational acceleration (9.81 m/s²) - MF microfilament - NIZEMI Niedergeschwindigkeits-Zentrifugen-Mikroskop (slow-rotating-centrifuge-microscope)     

Ultrastructural evidence for the presence of actin filaments in mouse eggs at fertilization

Summary Mouse eggs at fertilization were permeated with glycerol solutions and then reacted with heavy meromyosin to show actin filaments by electron microscopy. The meiotic area of the egg surface is devoid of microvilli and is supported by a thick layer (0.6–0.8 m in width) of submembranous filaments. A much thinner layer (less than 0.3 m) is present in the remaining non-meiotic microvillous area and underlying its membrane is a very thick layer of cross-filaments and filament bundles.     

Dynamic aspects of apical zonation in the angiosperm pollen tube

Summary In the apical 10–20 m of actively extending pollen tubes of Epilobium angustifolium, in a zone where the polysaccharide-containing wall precursor bodies (P-particles) dominate and where their movements on superficial observation seem to be random, there is in fact a concerted flux, acropetal movement taking place along the flanks of the tip zone, with a basipetal return flow along the centre. Detailed tracking of individuals shows that lipid globuli (diameters up to 1.5 m) and amyloplasts (dimensions up to 5.5 × 2.5 m) follow similar patterns of movement, but are sorted out in the sub-apical region, the smaller bodies penetrating further towards the apex. The findings are interpreted as indicating that the well-documented apical zonation of the pollen tube is maintained in the fluid circumstances of the growing tube by the filtering of cytoplasmic inclusions through the actin cytoskeleton, which, in conformity with recent fine-structural and other observations, is envisaged as consisting of a network of cross-linked microfilaments and microfilament aggregates at the tube tip giving place progressively to a system of more ordered, longitudinally oriented fibrils in the older parts of the tube. The implications for the operation of the actomyosin motility system and the tip growth mechanism are discussed.     

Micromanipulation of statoliths in gravity-sensing Chara rhizoids by optical tweezers

Infrared laser traps (optical tweezers) were used to micromanipulate statoliths in gravity-sensing rhizoids of the green alga Chara vulgaris Vail. We were able to hold and move statoliths with high accuracy and to observe directly the effects of statolith position on cell growth in horizontally positioned rhizoids. The first step in gravitropism, namely the physical action of gravity on statoliths, can be simulated by optical tweezers. The direct laser microirradiation of the rhizoid apex did not cause any visible damage to the cells. Through lateral positioning of statoliths a differential growth of the opposite flank of the cell wall could be induced, corresponding to bending growth in gravitropism. The acropetal displacement of the statolith complex into the extreme apex of the rhizoid caused a temporary decrease in cell growth rate. The rhizoids regained normal growth after remigration of the statoliths to their initial position 10–30 m basal to the rhizoid apex. During basipetal displacement of statoliths, cell growth continued and the statoliths remigrated towards the rhizoid tip after release from the optical trap. The resistance to statolith displacement increased towards the nucleus. The basipetal displacement of the whole complex of statoliths for a long distance (>100 m) caused an increase in cell diameter and a subsequent regaining of normal growth after the statoliths reappeared in the rhizoid apex. We conclude that the statolith displacement interferes with the mechanism of tip growth, i.e. with the transport of Golgi vesicles, either directly by mechanically blocking their flow and/or, indirectly, by disturbing the actomyosin system. In the presence of the actin inhibitor cytochalasin B the optical forces required for acropetal and basipetal displacement of statoliths were significantly reduced to a similar level. The lateral displacement of statoliths was not changed by cytochalasin B. The results indicate: (i) the viscous resistance to optical displacement of statoliths depends mainly on actin, (ii) the lateral displacement of statoliths is not impeded by actin filaments, (iii) the axially directed actin-mediated forces against optical displacement of statoliths (for a distance of 10 m) are stronger in the basipetal than in the acropetal direction, (iv) the forces acting on single statoliths by axially oriented actin filaments are estimated to be in the range of 11–110 pN for acropetal and of 18–180 pN for basipetal statolith displacements.Abbreviation CB cytochalasin B This work was supported by the Bundesminister für Forschung und Technologie, and by Fonds der Chemischen Industrie. We thank Professor Dr. A. Sievers (Botanisches Institut, Universität Bonn, Germany) for helpful discussions.     

Actin filament organization and polarity in pollen tubes revealed by myosin II subfragment 1 decoration

The actin cytoskeleton plays a crucial role in pollen tube growth. In elongating pollen tubes the organization and arrangement of actin filaments (AFs) differs between the shank and apical region. However, the orientation of AFs in pollen tubes has not yet been successfully demonstrated. In the present work we have used myosin II subfragment 1 (S1) decoration to determine the polarity of AFs in pollen tubes. Electron microscopy studies revealed that in the shank of the tube bundles of AFs exhibit uniform polarity with those close to the cell cortex having their barbed ends oriented towards the tip of the pollen tube while those in the cell center have their barbed ends oriented toward the base of the tube. At the subapex, some AFs are organized in closely packed and longitudinally oriented bundles and some form curved bundles adjacent to the cell membrane. In contrast, few AFs are dispersed with random orientation in the extreme apex of the pollen tube. Our results confirm that the direction of cytoplasmic streaming within pollen tubes is determined by the polarity of AFs in the bundles.     

Hypaphorine, an indole-3-acetic acid antagonist delivered by the ectomycorrhizal fungus Pisolithus tinctorius, induces reorganisation of actin and the microtubule cytoskeleton in Eucalyptus globulus ssp bicostata root hairs

Hypaphorine, an indole alkaloid from the ectomycorrhizal fungus Pisolithus tinctorius Coker & Couch., counteracts indole-3-acetic acid (IAA) activity and controls the rate of root hair elongation in Eucalyptus globulus ssp. bicostata. The present investigation shows that hypaphorine changes cytoskeletal organisation in elongating root hairs of the host. The actin cytoskeleton was investigated by two different fixation and labelling procedures, which gave similar results. In control root hairs, actin organisation was characterised by (i) an actin cap at the very tip region, (ii) a subapical region with reduced labelling and containing fine actin filaments, and (iii) axial bundles of actin filaments running from the subapical part to the base of the root hair. In the hypaphorine-treated root hairs no actin cap was distinguished. The fine actin filaments occurring in the subapical region were replaced by a few thick actin filament bundles that extended from the subapical region toward the root hair tip. In the hypaphorine-treated hairs the total number of actin filament bundles along most of the root hair length was significantly reduced, presumably due to aggregation of pre-existing actin filaments. The first signs of alteration to the cytoskeleton could be detected as soon as 15 min after hypaphorine treatment. In hypaphorine-treated, but not in control root hairs, a patch of aggregated microtubules regularly occurred at a distance of approximately 10 m from the tip, possibly as a consequence of changes induced by hypaphorine in the actin cytoskeleton. The hypaphorine-induced aggregations in the actin and microtubule cytoskeletons could stabilise the structure of cytoskeletal elements, which in turn could hinder the vesicle delivery at the tip necessary for elongation. Such cytoskeletal alterations may be a consequence of the antagonism between IAA and hypaphorine. The latter view was supported by restoration of the actin cytoskeleton in hypaphorine-treated root hairs by IAA application.     

Microfilament Orientation Constrains Vesicle Flow and Spatial Distribution in Growing Pollen Tubes

The dynamics of cellular organelles reveals important information about their functioning. The spatio-temporal movement patterns of vesicles in growing pollen tubes are controlled by the actin cytoskeleton. Vesicle flow is crucial for morphogenesis in these cells as it ensures targeted delivery of cell wall polysaccharides. Remarkably, the target region does not contain much filamentous actin. We model the vesicular trafficking in this area using as boundary conditions the expanding cell wall and the actin array forming the apical actin fringe. The shape of the fringe was obtained by imposing a steady state and constant polymerization rate of the actin filaments. Letting vesicle flux into and out of the apical region be determined by the orientation of the actin microfilaments and by exocytosis was sufficient to generate a flux that corresponds in magnitude and orientation to that observed experimentally. This model explains how the cytoplasmic streaming pattern in the apical region of the pollen tube can be generated without the presence of actin microfilaments.     

Inhibition of actin polymerisation by low concentration Latrunculin B affects endocytosis and alters exocytosis in shank and tip of tobacco pollen tubes

Pollen tube growth depends on the integrity of the actin cytoskeleton that regulates cytoplasmic streaming and secretion. To clarify whether actin also plays a role in pollen tube endocytosis, Latrunculin B (LatB) was employed in internalisation experiments with tobacco pollen tubes, using the lipophilic dye FM4‐64 and charged nanogold. Time‐lapse analysis and dissection of endocytosis allowed us to identify internalisation pathways with different sensitivity to LatB. Co‐localisation experiments and ultrastructural observations using positively charged nanogold revealed that LatB significantly inhibited endocytosis in the pollen tube shank, affecting internalisation of the plasma membrane (PM) recycled for secretion, as well as that conveyed to vacuoles. In contrast, endocytosis of negatively charged nanogold in the tip, which is also conveyed to vacuoles, was not influenced. Experiments of fluorescence recovery after photobleaching (FRAP) of the apical and subapical PM revealed domains with different rates of fluorescence recovery and showed that these differences depend on the actin cytoskeleton integrity. These results show the presence of distinct degradation pathways by demonstrating that actin‐dependent and actin‐indepedent endocytosis both operate in pollen tubes, internalising tracts of PM to be recycled and broken down. Intriguingly, although most studies concentrate on exocytosis and distension in the apex, the present paper shows that uncharacterised, actin‐dependent secretory activity occurs in the shank of pollen tubes.     

The cytoskeleton of the fiber cells of Trichoplax adhaerens (Placozoa)

Summary The cytoskeleton of Trichoplax adhaerens fiber cells was studied after chemical fixation, freeze-substitution, lysis of attached cells with nonionic detergents and by immunofluorescence. Cytoskeletal elements present in the cell bodies and reaching into the extensions include microtubules, intermediate filaments, 6–7 nm and 2–3 nm microfilaments. The latter seem to interconnect other cytoskeletal elements. Actin-like microfilaments are found both as networks and parallel strands. Immunofluorescence with antiactin shows the presence of actin in the cell body, underneath the plasmalemma and within the extensions. Both the results of immunofluorescence and the identification of 6–7 nm actin-like microfilaments support the concept of contractility of the fiber cells as the cause of the rapid shape changes of Trichoplax. Anti-tubulin fluorescence corresponds to the location of microtubules in the extensions as well as the cell bodies of the fiber cells. The extensions are withdrawn upon depolymerization of the microtubules by colchicine.     

FIMBRIN1 Is Involved in Lily Pollen Tube Growth by Stabilizing the Actin Fringe

An actin fringe structure in the subapex plays an important role in pollen tube tip growth. However, the precise mechanism by which the actin fringe is generated and maintained remains largely unknown. Here, we cloned a 2606-bp full-length cDNA encoding a deduced 77-kD fimbrin-like protein from lily (Lilium longiflorum), named FIMBRIN1 (FIM1). Ll-FIM1 was preferentially expressed in pollen and concentrated at actin fringe in the subapical region, as well as in longitudinal actin-filament bundles in the shank of pollen tubes. Microinjection of Ll-FIM1 antibody into lily pollen tubes inhibited tip growth and disrupted the actin fringe. Furthermore, we verified the function of Ll-FIM1 in the fim5 mutant of its closest relative, Arabidopsis thaliana. Pollen tubes of fim5 mutants grew with a larger diameter in early stages but could recover into normal forms in later stages, despite significantly slower growth rates. The actin fringe of the fim5 mutants, however, was impaired during both early and late stages. Impressively, stable expression of fim5pro:GFP:Ll-FIM1 rescued the actin fringe and the growth rate of Arabidopsis fim5 pollen tubes. In vitro biochemical analysis showed that Ll-FIM1 could bundle actin filaments. Thus, our study has identified a fimbrin that may stabilize the actin fringe by cross-linking actin filaments into bundles, which is important for proper tip growth of lily pollen tubes.     

Morphological evidence for the existence of a more complex cytoskeleton in Amoeba proteus

Summary Various stabilization and extraction procedures were tested to demonstrate the ultrastructural organization of the cytoskeleton in normal, locomoting Amoeba proteus. Most reliable results were obtained after careful fixation in glutaraldehyde/lysine followed by prolonged extraction in a polyethylene glycol/Triton X-100 solution. Before dehydration in a graded series of ethanol and critical-point drying, the amoebae were split by the sandwich-technique, i.e., by mechanical cleavage of cells mounted between two poly-L-lysine-coated glass slides. Platinum-carbon replicas as well as thin sections prepared from such cell fragments revealed a cytoskeleton composed of at least four different types of filaments: (1) 5–7-nm filaments organized as a more or less ordered cortical network at the internal face of the plasma membrane and probably representing F-actin; (2) 10–12-nm filaments running separately or slightly aggregated through the cytoplasm and probably representing intermediate filaments; (3) 24–26-nm filaments forming a loose network and probably representing microtubules; and (4) 2–4-nm filaments as connecting elements between the other cytoskeleton constituents. Whereas microfilaments are responsible for protoplasmic streaming and other motile phenomena, the function of intermediate filaments and cytoplasmic microtubules in amoebae is still obscure.     

Actin microfilaments do not form a dense meshwork inLilium longiflorum pollen tube tips

Summary Controversy over whether the apical region of a growing pollen tube contains a dense array of actin microfilaments (MFs) was the impetus for the present study. Microinjection of small amounts of fluorescently labeled phalloidin allowed the observation of MF bundles inLilium longiflorum pollen tubes that were growing and functioning normally. The results show that while the pollen tube contains numerous MF bundles arranged axially, the apical region is essentially devoid of them. The MF bundles could be seen shifting and changing in distribution as the cells grew, but they always remained out of the apical regions. Perturbation of normal growth and function by caffeine causes a change in the MF distribution, which returns to normal upon removal of caffeine from the growth medium. The lack of MFs in the apex is confirmed by careful immunogold electron microscopic analysis of thin sections of rapidly frozen and freeze-substituted pollen tubes, in which very fine MF bundles could be seen somewhat closer to the tip than is discernible with fluorescence microscopy. Still, these are very few in number and are basically absent from the very tip. Thus a reassessment of current assumptions about the distribution of actin in the pollen tube apical region is required.Abbreviations MF microfilaments - FITC fluorescein isothiocyanate - RF-FS rapidly frozen and freeze-substituted - EM electron microscopy Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Effect of tree distance on N2O and CH4-fluxes from soils in temperate forest ecosystems

Complete annual cycles of N₂O and CH₄ flux in forest soils at a beech and at a spruce site at the Höglwald Forest were followed in 1997 by use of fully automatic measuring systems. In order to test if on a microsite scale differences in the magnitude of trace gas exchange between e.g. areas in direct vicinity of stems and areas in the interstem region at both sites exist, tree chambers and gradient chambers were installed in addition to the already existing interstem chambers at our sites. N₂O fluxes were in a range of –4.6–473.3 g N₂O-N m^–2 h^–1 at the beech site and in a range of –3.7–167.2 g N₂O-N m^–2 h^–1 at the spruce site, respectively. Highest N₂O emissions were observed during and at the end of a prolonged frost period, thereby further supporting previous findings that frost periods are of crucial importance for controlling annual N₂O losses from temperate forests. Fluxes of CH₄ were in a range of +10.4––194.0 g CH₄ m^–2 h^–1 at the beech site and in a range of –4.4––83.5 g CH₄ m^–2 h^–1 at the spruce site. In general, both N₂O-fluxes as well as CH₄-fluxes were higher at the beech site. On a microsite scale, N₂O and CH₄ fluxes at the beech site were highest within the stem area (annual mean: 49.6±3.3 g N₂O-N m^–2 h^–1; –77.2±3.1 g CH₄ m^–2 h^–1), and significantly lower within interstem areas (18.5±1.4 g N₂O-N m^–2 h^–1; –60.2±1.8 g CH₄ m^–2 h^–1). Significantly higher values of total N, C and pH in the organic layer, as well as increased soil moisture, especially in spring, in the stem areas, are likely to contribute to the higher N₂O fluxes within the stem area of the beech. Also for the spruce site, such differences in trace gas fluxes could be demonstrated to exist (mean annual N₂O emission within (a) stem areas: 9.7±0.9 g N₂O-N m^–2 h^–1 and (b) interstem areas: 6.2±0.6 g N₂O-N m^–2 h^–1; mean annual CH₄ uptake within (a) stem areas: –26.1±0.6 g CH₄ m^–2 h^–1 and (b) interstem areas: –38.4±0.8 g CH₄ m^–2 h^–1), though they were not as pronounced as at the beech site.     

The cytoskeleton of Cobaea seed hairs:

The cell wall of Cobaea scandens seed hairs developed in a characteristic sequence, with the deposition of a cellulose thread onto a pectic swelling layer was the final event. The cellulose thread was intracellularly accompanied by a band of 10–18 microtubules. During the formation of the swelling layer the microtubules were homogeneously distributed; they ran circumferentially normal to the cell axis. When cellulose-thread formation started, the microtubules became arranged in a helical band. The density of the microtubules varied during the different phases of development. The highest density was observed before cellulosethread formation and ranged from 6–15 m·m^-2. The length of the microtubules, 20–30 m, was determined by direct measurements, as well as estimated from the total microtubular length in a given area and the counted free ends. With the indirect immunofluorescence technique the microtubules of the band stained inhomogeneously. Those which were located at the edges of the band fluoresced more intensely than those of the central part. Attempts to visualize actin filaments in the hair cells with rhodaminyl-conjugated phalloidin resulted in a homogeneous staining of the area of the microtubular band, indicating that actin filaments may be present in this region. Though, in thin sections and dry-cleaved cells, filamentous structures were observed between the microtubules, caution is expressed that the observed fluorescence was, indeed, due to actin filaments. The role of the filamentous structures is discussed with respect to formation and maintenance of the microtubular band. Microtubules apparently did not cross coated pits which were visualized in the plasma membrane through the dry-cleaving technique.Abbreviations IFT indirect immunofluorescence technique - RP rhodaminyl-conjugated phalloidin - SEM scanning electron microscopy