Characterization of endoreplicated nuclei in Arabidopsis thaliana

A model plant, Arabidopsis thaliana, duplicates its chromosomes without cell division, in a process known as endoreplication. The primary objective of this study was to identify genes and proteins that specifically accumulate in endoreplicated nuclei in Arabidopsis thaliana. Analysis of nuclei sorted by fluorescent activated cell sorter (FACS) revealed that ploidy levels and nuclear volume were positively correlated. However, the protein amounts among 2C, 4C, and 8C nuclei had no significant differences. This indicated a decrease of the protein concentration in endoreplicated nuclei. In contrast, the RNA amount in the 8C nuclei was 1.3- and 1.4-fold higher than that in the 4C and 2C nuclei, respectively. This suggested an increase of RNA after the second round of endoreplication. The secondary objective was to identify the biological function of unique domains found in Arabidopsis topoisomerase VI subunit B (AtTopVIB) that contributes to endoreplication. Using the AtTopVIB amino acid sequence and protein database search engine, I identified two unique domains to which I designated the insertion of the N-terminal domain (IND) and the extension in the C-terminal domain (ECD). These domains are well conserved between Arabidopsis and Oryza sativa (rice) but very unique in the entire family. Towards understanding the biological function of these domains, I analyzed the localization of AtTopVIB in Arabidopsis protoplasts with yellow-fluorescent protein (YFP). The results indicated that AtTopVIB was localized in the nucleus. Also, I analyzed AtTopVIB self interaction in Arabidopsis protoplasts using split-luciferase. The results indicated a weak self interaction of AtTopVIB. These results suggested that the IND and ECD may be involved in the localization or self-interaction of AtTopVIB in Arabidopsis cells.