Chromatin alterations during pollen development in Hordeum vulgare.

The dynamics of posttranslational histone modifications in relation to nuclear architecture has been analyzed during pollen development in Hordeum vulgare L. cv. Igri. Notwithstanding the asymmetry of cytokinesis associated with pollen mitosis I, immunolabeling revealed that the vegetative and generative nuclei initially display identical chromatin modification patterns. Yet, differential chromatin modification patterns between vegetative and generative nuclei emerge with the development of conspicuous differences in nuclear morphology as visualized by 4',6-diamidino-2-phenylindole staining. The temporal and spatial distribution of most histone modifications observed is in agreement with reduced gene activity in the generative nucleus and increased expression in the vegetative nucleus as indicated by immunolabeling of active RNA polymerase II. Signals of trimethylation of histone H3 lysine 27 proved to be particularly enriched in euchromatic domains of subtelomeric regions. In the context of nuclear differentiation in bicellular pollen, this modification became restricted to the vegetative nucleus, indicating a role in activating rather than suppressing gene expression. The presence of acetylated histone H3 at lysine 9 in the cytoplasm of the generative cell is indicative of a more complex, still unknown function of this particular modification.

Time-lapse imaging of the initiation of pollen embryogenesis in barley (Hordeum vulgare L.).

Pollen embryogenesis provides exciting opportunities in the areas of breeding and biotechnology as well as representing a convenient model for studying the process of plant cell proliferation in general and embryogenesis in particular. A cell culture system was devised in which immature barley pollen could be cultured as a monolayer trapped between the bottom glass-cover slip of a live-cell chamber and a diaphanous PTFE membrane within a liquid medium over a period of up to 28 d, allowing the process of embryogenesis to be tracked in individual pollen. Z-stacks of images were automatically captured every 3min, starting from the unicellular pollen stage up to the development of multicellular, embryogenic structures. The method should prove useful for the elucidation of ultrastructural features and molecular processes associated with pollen embryogenesis.

The use of cyanobacteria as filler in nitrocellulose capillaries improves ultrastructural preservation of immature barley pollen upon high pressure freezing.

The high pressure freezing (HPF) followed by freeze substitution technique has advantages over chemical fixation in the context of preserving sample ultrastructure. However, when HPF is applied to cultured pollen grains, the large intercellular spaces present lead to a poor level of ultrastructure preservation. We report here that the mixing of cyanobacteria with immature barley pollen grains succeeded in greatly reducing the volume of liquid present between the large pollen grains, and so improved the loading of the sample into a nitrocellulose capillary. The use of yeast or cyanobacteria paste to surround the filled capillaries was beneficial in speeding the transfer of heat during the freezing process. This modification of the HPF method resulted in a greatly improved level of ultrastructure preservation.