Changes in Nuclear Structure During Wheat Endosperm Development

This thesis is an investigation into the structure of wheat endosperm nuclei starting with nuclear divisions and migration during syncytium formation followed by the development of nuclear shape and positioning of chromosome territories and ending with changes in subchromosomal structure during the activation of a transgene locus.<o:p></o:p></span>At the level of the whole endosperm we have developed a method for modelling in 3D the formation of the syncytium that characterises early endosperm development. After the initial nuclear division of the first endosperm nucleus three groups of nuclei form in the original central cell: a stem-like group of nuclei close to the zygote and connected to a single layer of nuclei in the dorsal periphery and another, unconnected single layer in the ventral periphery. By two days post anthesis (dpa) both the ventral and dorsal groups of nuclei have developed into plates of nuclei. The dorsal plate then merges with the ventral plate through synchronous nuclear divisions. By 4 dpa the entire periphery of the central cell is surrounded by a layer of nuclei and the syncytium is complete.<o:p></o:p></span>Wheat endosperm is initially triploid and during its development a percentage of the nuclei increase their DNA-content to 6C and 12C. 3D modelling of nuclei with DNA contents of 3C, 6C and 12C allowed us to visualise progressive changes in nuclear shape and chromosome positioning. With increasing C content, nuclear volumes increase predominantly in two directions, thereby changing the shape of the nuclei into a disc-like structure. Wheat chromosomes in interphase nuclei have a typical Rabl configuration with centromeres and telomeres arranged at opposite poles. The majority of centromeres and telomeres are found at or close to the nuclear membrane, some also in the middle of the nucleus and in rare instances a telomere can be observed at the centromere pole and vice versa.<span style='mso-spacerun:yes'> </span>This means that centromeres and telomeres are not or only transiently anchored to the nuclear membrane. Both centromeres and telomeres show a degree of non-homologous associations, which for centromeres remains constant through increases in ploidy, while telomere associations increase with higher C-values.<o:p></o:p></span>The wheat line we used has a 1RS / 1BS ( Secale cereale ) chromosome arm substitution. Fluorescence in situ hybridisation detection of the rye arm substitution with total genomic DNA as probe revealed the following: during endosperm development chromosomes are able to change their position in all three dimensions and as the C-content increases in nuclei that have stopped dividing chromosome arrangements become progressively distorted. The vast majority of 12 C nuclei show six rye chromosome arms, but a few show three groups of associated rye chromosome arms. This means that during endosperm development wheat nuclei increase their ploidy through both polyploidisation and polytenisation.<o:p></o:p></span>The final part analyses the chromatin structure of active and inactive gene loci. The High Molecular Weight (HMW) glutenin genes in wheat are developmentally activated in the endosperm at about 8 dpa. I have investigated the physical changes that occur in these genes in two transgenic lines containing about 20 and 50 copies respectively of the HMW glutenin genes together with their promoters. Fluorescence in situ hybridisation (FISH) and confocal imaging data show that in non-expressing tissue each transgene locus consists of one or two highly condensed sites that decondense into many foci upon activation of transcription in endosperm nuclei. Initiation of transcription can precede decondensation but not vice versa. In one of the lines, cytoplasmic transcript levels are high after onset of transcription, but disappear by 14 dpa, while siRNAs, indicative of post-transcriptional gene silencing (PTGS), are detected at this stage. However, the transcript levels remain high at the transcription sites, the great majority of the transgene copies are transcriptionally active and transcriptional activity in the nucleus ceases only with cell death at the end of endosperm development