PO11 dependent initiation of meiotic double strand breaks in <em>Arabidopsis thaliana</em>

Abstract

The exchange and reorganization of genetic material between two individuals is an essential function of meiosis. Even though in the last years major findings in the field of meiosis have been achieved, especially in plants some key questions remain concealed. For a better understanding of plant meiosis it is essential to decipher why plants need the activity of two very different meiotic SPO11 proteins. This stands in contrast to metazoa and fungi where a single SPO11 is present and sufficient for a proper meiosis. In Arabidopsis thaliana two SPO11, referred to as Ath SPO11-1 and Ath SPO11-2, are necessary in a functional form for the induction of double strand breaks (DSBs) during prophase I of meiosis. In nearly all eukaryotic organisms DSBs ensure on one hand the overall genome stability by enabling correct pairing and distribution of the chromosomes and on the other hand genetic variability by permitting recombination. Without DSBs no physical connection can occur between homologous chromosomes and recombination, pairing, and crossing over are excluded. The absence of these processes leads to a random distribution of the chromosomes during meiosis and to almost complete sterility. Therefore, the evolution, the specific functions, and possible interaction of both different meiotic SPO11 proteins in plants have been analyzed in this thesis. For this purpose database searches were performed and homologs of SPO11 in nearly all kingdoms of life were identified and analyzed. To determine the specific functions of both SPO11 paralogs in A. thaliana three non-conserved parts and the well conserved last exon between both meiotic SPO11 were exchanged. Furthermore, the later one was completely deleted, creating chimeric SPO11 genes. Analyzing these exchanges a sequence specific function for both SPO11 proteins was shown. By exchanging Ath SPO11-1 and -2 with related and more ancestral SPO11, additionally, species specificity for the respective SPO11 was exposed. Complementation was only possible using SPO11 from the close related (20 mya) species Brassica rapa. Partial complementation was achieved when SPO11-1 cDNA from papaya (Carica papaya) was integrated multiple times. By performing these analyses a vast pattern of aberrant spliced forms for SPO11-1 and -2 in various species were identified. By examination of the splicing landscape of orthologs from both SPO11 in its respective source plant and after transformation into A. thaliana, furthermore, a species and sequence specificity for the splicing process of SPO11 have been revealed. Due to analyses performed on the splicing landscape of the chimeric SPO11 genes a sequence specific distribution of splicing events was identified. These findings indicate that the function and the conserved splicing mechanism of each meiotic active SPO11 paralog is sequence specific and that function of the respective orthologs are species specific. To investigate a possible interaction of both SPO11 during meiosis an antibody against SPO11-2 was produced and a co-immunolocalization study for SPO11-1 and -2 was performed. First findings illustrate that both SPO11 paralogs seem to colocalize during early prophase I. However, SPO11-2 seems to stay longer on the chromatin.