Monitoring of nuclear activity during pollen development and androgenesis in Brassica napus L.

To study transcription and maturation of RNA, pollen development and androgenesis of Brassica napus was chosen as a test system as explained in Chapter 1. Culturing microspores and young bicellular pollen at 32 °C allows to change the developmental pathway from pollen development towards embryogenesis. Culturing at 18 °C results in normal pollen development.The ultrastructure of the nucleus was analyzed during pollen development applying various staining techniques which visualised the degree of chromatin condensation, the appearance of the interchromatin region and the nucleolar structure. The interchromatin region contains proteins which are necessary for the transcription and maturation of RNA.The results give a rough indication about the transcriptional activity at various pollen stages. Although difficult to interpret, they indicate an increase in transcription activity in nuclei from the young microspore stage up to the vegetative cell in almost mature pollen. The generative nucleus in early pollen initially showed all signs of a transcriptionally active nucleus, but it lost its transcriptionally active morphology prior to pollen mitosis. After pollen mitosis the chromatin of the sperm nuclei remained condensed, indicating that the sperm nuclei are transcriptionally silenced.To get more insight into the cell cycle stages of the various nuclei of bicellular and tricellular pollen and under embryogenic and non-embryogenic culture conditions, the DNA synthesis was analysed using bromodeoxyuridine. It was found that microspores are able to change their developmental pathway towards embryogenesis from G1 upto G2. In bicellular pollen the vegetative nucleus is arrested in the G1 and re-enters the cell cycle after embryo induction (Binarova et al., Theor. Appl. Genet. 87 : 9 - 16).To visualise RNA synthesis in plant nuclei in detail, procedures were developed to label the sites of transcription using the uridine-analogue 5'-bromouridine 5'-triphosphate (BrUTP). It is assessed that this method works well on isolated leaf cell nuclei of Brassica napus . It enabled to visualize transcription sites throughout the plant nucleus like was shown before in nuclei of vertebrate cells (Straatman et al., Protoplasma 192 : 145 - 149).The method was further developed, using leaf and pollen protoplasts, and germinating pollen with pollen tubes. In pollen protoplasts and pollen tubes, labelling of RNA transcription was only found in the vegetative nucleus; the sperm nuclei were silenced, which is in agreement with the ultrastructural analysis. It was further found that transcription in the vegetative nucleus of mature pollen protoplasts could be very intense whereas the transcription of this nucleus was relatively low in pollen tubes. However, attempts to label transcription sites during pollen development and after embryo-induction did not result in any signal.Leaf and pollen protoplasts were also used to analyse the maturation of pre-mRNA and to understand the relation between the transcription sites and the location of small nuclear ribonucleoprotein particles (snRNPs) and small nucleolar RNPs (snoRNPs). The snRNP distribution was also studied during pollen development and androgenesis. snRNPs were not detected in the generative nucleus of late bicellular pollen and in the sperm nuclei of tricellular pollen. This is in accordance with the ultrastructural analysis of these stages where the chromatin was more condensed in late bicellular pollen than in earlier stages indicating a low or silenced transcription. In sperm cells the chromatin was even more condensed and no incorporation of BrUTP was detected. However, also the vegetative nucleus of mature pollen did not label with antibodies against snRNPs, although the chromatin was very dispersed which points to a high transcriptional activity, as was found in the pollen protoplasts using BrUTP.A coiled body is one of the subcompartments in the nucleus containing snRNPs and fibrillarin. In general, the number of coiled bodies in nuclei of mammalian cells increases with an increase in transcriptional activity. Although there was an increase in the number of coiled bodies, the increase in size of these bodies was much more dramatic during pollen development. After embryo induction a slow decrease in size of the coiled bodies was found.All RNA leaving the nucleus and all nuclear proteins produced in the cytoplasm have to pass the nuclear pore complexes (NPCs). Therefore, the NPC density can give an indication about general nuclear activity. During pollen development the NPC density slowly increased and reached its highest level in the vegetative nucleus of mature pollen. This correlates well with the fact that just before the vegetative nucleus is silenced, the transcription is high in this nucleus. The NPC density of the sperm nuclei was low which confirms the state of low activity as has been described. After or during embryo induction a decrease in NPC density was found. However, this occurred under both embryogenic and non-embryogenic conditions, indicating that it is not evidence for embryo induction.</p

Mitotic phosphorylation by NEK6 and NEK7 reduces microtubule affinity of EML4 to promote chromosome congression

EML4 is a microtubule-associated protein that promotes microtubule stability. We investigated its regulation across the cell cycle and found that EML4 was distributed as punctate foci along the microtubule lattice in interphase but exhibited reduced association with spindle microtubules in mitosis. Microtubule sedimentation and cryo-electron microscopy with 3D reconstruction revealed that the basic N-terminal domain of EML4 mediated its binding to the acidic C-terminal tails of α- and β-tubulin on the microtubule surface. The mitotic kinases NEK6 and NEK7 phosphorylated the EML4 N-terminal domain at Ser144 and Ser146 in vitro, and depletion of these kinases in cells led to increased EML4 binding to microtubules in mitosis. An S144A-S146A double mutant not only bound inappropriately to mitotic microtubules but also increased their stability and interfered with chromosome congression. Meanwhile, constitutive activation of NEK6 or NEK7 reduced EML4 association with interphase microtubules. Together, these data support a model in which NEK6- and NEK7- dependent phosphorylation promotes dissociation of EML4 from microtubules in mitosis in a manner that is required for efficient chromosome congression

Distribution of splicing proteins and putative coiled bodies during pollen development and androgenesis in Brassica napus L.

Small nuclear ribonucleoprotein particles (snRNPs) are subunits of splicing complexes, which show a transcription-dependent localization pattern. We have analyzed the labelling pattern of snRNPs during pollen development and microspore and pollen embryogenesis in Brassica napus with an antibody which recognizes protein D of U1, U2, U4, U5, and U6 snRNPs. It was found that nuclei were labelled almost uniformly for snRNPs in microspores and young bicellular pollen. In the generative nuclei of late-bicellular pollen and in the vegetative nuclei and sperm nuclei of mature pollen no snRNPs could be detected. The snRNP-positive nuclei contained mostly one or two brightly labelled nuclear bodies, most likely coiled bodies, often closely related to the nucleolus. These nuclear bodies increased in size from 0.5 micron in nuclei of young microspores up to 2 microns in nuclei of late microspores and the vegetative nucleus of early-bicellular pollen. Also their number increased during these developmental stages. After induction of embryogenesis the size of the coiled bodies decreased to about 0.5 micron and in several occasions the coiled body was found free in the nucleoplasm, away from the nucleolus. The results support the idea that the size and number of coiled bodies coincide with changes in general nuclear activity. They also indicate that, in nuclei of Brassica napus, at least assembly and disassembly of coiled bodies takes place in the nucleoplasm, whereas mature coiled bodies are located adjacent to the nucleolus