Gametophytic development of Brassica napus pollen in vitroenables examination of cytoskeleton and nuclear movements

Isolated microspores and pollen suspension of Brassica napus “Topas” cultured in NLN-13 medium at 18°C follow gametophytic pathway and develop into pollen grains closely resembling pollen formed in planta. This culture system complemented with whole-mount immunocytochemical technology and novel confocal laser scanning optical technique enables detailed studies of male gametophyte including asymmetric division, cytoskeleton, and nuclear movements. Microtubular cytoskeleton configurationally changed in successive stages of pollen development. The most prominent role of microtubules (MTs) was observed just before and during nuclear migration at the early and mid-bi-cellular stage. At the early bi-cellular stage, parallel arrangement of cortical and endoplasmic MTs to the long axis of the generative cell (GC) as well as MTs within GC under the plasmalemma bordering vegetative cell (VC) were responsible for GC lens shape. At the beginning of the GC migration, endoplasmic microtubules (EMTs) of the VC radiated from the nuclear envelope. Most cortical and EMTs of the VC were found near the sporoderm. At the same time, pattern of MTs observed in GC was considerably different. Multiple EMTs of the GC, previously parallel aligned, reorganized, and start to surround GC, forming a basket-like structure. These results suggest that EMTs of GC provoke changes in GC shape, its detachment from the sporoderm, and play an important role in GC migration to the vegetative nucleus (VN). During the process of migration of the GC to the VC, multiple and thick bundles of MTs, radiating from the cytoplasm near GC plasma membrane, arranged perpendicular to the narrow end of the GC and organized into a “comet-tail” form. These GC “tail” MTs became shortened and the generative nucleus (GN) took a ball shape. The dynamic changes of MTs accompanied polarized distribution pattern of mitochondria and endoplasmic reticulum. In order to confirm the role of MTs in pollen development, a “whole-mount” immunodetection technique and confocal laser-scanning microscopy was essential

Microtubule configurations and nuclear DNA synthesis during initiation of suspensor-bearing embryos from Brassica napus cv. Topas microspores

In the new Brassica napus microspore culture system, wherein embryos with suspensors are formed, ab initio mimics zygotic embryogenesis. The system provides a powerful in vitro tool for studying the diverse developmental processes that take place during early stages of plant embryogenesis. Here, we studied in this new culture system both the temporal and spatial distribution of nuclear DNA synthesis places and the organization of the microtubular (MT) cytoskeleton, which were visualized with a refined whole mount immunolocalization technology and 3D confocal laser scanning microscopy. A ‘mild’ heat stress induced microspores to elongate, to rearrange their MT cytoskeleton and to re-enter the cell cycle and perform a predictable sequence of divisions. These events led to the formation of a filamentous suspensor-like structure, of which the distal tip cell gave rise to the embryo proper. Cells of the developing pro-embryo characterized endoplasmic (EMTs) and cortical microtubules (CMTs) in various configurations in the successive stages of the cell cycle. However, the most prominent changes in MT configurations and nuclear DNA replication concerned the first sporophytic division occurring within microspores and the apical cell of the pro-embryo. Microspore embryogenesis was preceded by pre-prophase band formation and DNA synthesis. The apical cell of the pro-embryo exhibited a random organization of CMTs and, in relation to this, isotropic expansion occurred, mimicking the development of the apical cell of the zygotic situation. Moreover, the apical cell entered the S phase shortly before it divided transversally at the stage that the suspensor was 3–8 celled

Bleeding disease of horse chestnut and biogenesis of a structural barrier after wounding.

<p>(A) Typical symptoms of <i>P. syringae</i> pv. <i>aesculi</i> associated bleeding disease observed on the trunk of an <i>A. carnea</i> tree, including bleeding of amber coloured sap and cracking of the bark (arrowheads). Photo taken in Sept. 2008 at N51° 57′ 26″; E5° 34′ 10″. (B) Appearance of surface wounds on <i>A. hippocastanum</i> seedlings mock-inoculated (top panel) or inoculated with <i>P. s.</i> pv. <i>aesculi</i> PD5126 (bottom panel) after 3 months. While the seedling bark recovers when mock-inoculated by regeneration of periderm, the wounded tissue appears blackened and sunken when inoculated with <i>P. s.</i> pv. <i>aesculi</i>. (C) Immunofluorescent labelling of a transverse section of the wound area sampled directly after inoculation of <i>P. s.</i> pv. <i>aesculi</i> PD4818 (t = 0) indicates that the bacteria (green) only colonize the outermost cell layer after inoculation. The scale bar indicates 100 µm. (D) A longitudinal section of a mock-inoculated wound sampled after 6 days with phloroglucinol-HCl stained lignin (red/purple). A barrier zone composed of several lignified parenchyma cells wide is visible along the wound (arrowhead) along with a broad layer of diffusely lignified cells in the disjointed part (arrow). The scale bar indicates 0.5 mm. (E) Fluorescence microscopic observation on a longitudinal section of a wound sampled 8 days after PD4818 inoculation stained for waxes using Sudan IV. The cells that exhibit lignification, as seen by the yellow/green autofluorescence, also show Sudan IV stained suberin lamellae that appear in dim red. The arrow reflects the direction of the original inoculation cut. The scale bar indicates 50 µm. (F) Detail of E showing the even deposition of suberin around the plant protoplasts. The scale bar indicates 25 µm. (G) Continuous deposition of suberin by two cells at pit fields (arrowheads). The scale bar indicates 10 µm.</p

Bacterial strains and plasmids used in this study.

a<p>Abbreviations used: Cb, carbenicillin; Km, Kanamycin; Tc, Tetracycline; Tra, conjugal transfer functions.</p

A fibrillar matrix produced <i>in vitro</i> by <i>P. syringae</i> pv. <i>aesculi</i> enwraps aggregates of immobilized bacteria.

<p>(A) After incubation in minimal medium a fibrillar material covering clustered bacterial cells is observed by scanning electron microscopy (left-hand panel). Such material is not encountered after incubation in LB, wherein only the polar flagella are observed extracellularly (right-hand panel). The insets show the bacterial cells and the surrounding material in more detail. Note that due to the completely dehydrated state of the specimen, the here presented image likely does not depict the true spatial lay-out of a bacterial cluster. The scale bars indicate 1 µm. (B) A typical cluster observed in a PD4818-pMP4655 culture grown in minimal medium visualized by confocal laser scanning microscopy. GFP-expressing bacteria appear in green, while addition of propidium iodide (PI) brightly stained dead individuals red and led to a mild staining of the amorphous material enwrapping the bacteria within a cluster. The bottom and right panels are computed images of the two orthogonal planes along the z-axis indicated by the dark grey arrows. The light grey arrows indicate the position of the optical plane shown in the top left panel. Note that single bacteria are spaced from neighbouring cells in 3 dimensions. The scale bars indicate 10 µm. (C) Three 10 second interval frames taken from a time-lapse movie of the edge of a PD4818-pMP4655 cluster (depicted in the DIC image) were differentially colour coded and merged to reveal displacement of individual bacteria over time. Several motile bacteria freely moved on the left appearing in a single colour in the merged image, while bacteria within the cluster remained at a fixed location as revealed by their white colour in the merged image. Additionally, oscillatory motion of 2 bacteria is discernable in the cluster margin (arrowhead), indicated by the slight colour shift. The scale bar indicates 5 µm.</p

Effect of heat-treatment on recovery of bacteria from lesions in inoculated <i>A. hippocastanum</i> saplings.

a<p>The figures are given as number of successful re-isolations per no. of lesions assessed.</p

Behaviour of GFP-expressing <i>P. syringae in planta.</i>

<p>(A) An <i>A. hippocastanum</i> seedling inoculated with strain PD4818-pMP4655 photographed after 2 months shows vertical lesion expansion (up to 6 cm in length) and tissue necrosis after removal of the outer tissues of the bark. The inoculation site is indicated with an arrowhead. Note the bending of the infection around the lower node. The scale bar indicates 1 cm. (B) Side view of a longitudinally split piece of stem from a PD4818-pMP4655 inoculated seedling removed 1–2 cm from the inoculation site 4 weeks after inoculation. Necrosis and discolouration is visible in the cambial and phloem areas. (C) A longitudinal section of an infection border in bark tissue after inoculation with PD4818-pMP4655 sampled 4 weeks after inoculation. A parabolic shaped zone with elevated cell wall autofluorescence is discernible (arrowheads) with a <i>P. s.</i> pv. <i>aesculi</i> colony located in the tip of the infected zone, at about 1 cm away from the inoculation site. The boxed area is shown in detail in D. The scale bar indicates 25 µm. (D) Numerous bacteria are clustered near the forefront of an advancing lesion in parenchyma 4 weeks after inoculation. The bacteria observed here are relatively small and densely packed. The scale bar indicates 10 µm. (E) Within the necrotic area near the original site of inoculation several clusters of bacteria are found that occupy the cavities left by dead cells. Image taken from a longitudinal section of a 6 week old infection. The scale bar indicates 25 µm. (F) A large aggregate of PD4818-pMP4655 located in necrotic tissue of a 4 week old lesion. Individual bacteria were spaced apart and did not undergo any type of motility. The scale bar indicates 25 µm. (G) Detail of a PD4818-pMP4655 cluster in necrotic tissue of a 4 week old infection, emphasizing the spacing between individual bacteria. The scale bar indicates 10 µm. (H) Immunodetection of wild-type PD4818 in the necrotic tissue of a 10 week old infection showing a similar spatial arrangement of bacteria to that shown in G. The scale bar indicates 10 µm.</p