Dissecting the molecular function and regulation of the meiotic α-kleisins REC8 and RAD21.2 in Arabidopsis thaliana

Abstract

The faithful transmission of chromosomes during cell divisions is essential for the survival of eukaryotic organisms. Aside from mitosis, which leads to two genetically identical daughter cells, meiosis is required for sexual reproduction and ensures biodiversity via the formation of haploid gametes with newly assorted allelic combinations. Essential for a proper chromosome segregation during both cell divisions is the chromosomally bound multi-subunit complex called cohesin. The mitotic and meiotic complexes are formed by the heterodimer of the STRUCTURAL MAINTENANCE proteins SMC1 and SMC3. The binding of a α-kleisin protein to SMC1 and SMC3 closes and forms a ring shaped complex, keeping sister chromatids together. In Arabidopsis thaliana, four α-kleisins (RAD21.1, RAD21.2, RAD21.3 and REC8) have been identified so far. Although a function in vegetative cells has been postulated for the three RAD21 proteins, less is known about their exact roles. In contrast, the meiotic specific α-kleisin REC8 has been intensively studied revealing its requirement for proper chromosome segregation via its stepwise loss during meiosis. This was found to be dependent on a tightly controlled phosphorylation status of REC8 in organisms like yeast. However, the kinase responsible for the REC8 phosphorylation remains unknown in planta. A previous in vitro analysis identified CDKA;1 as a possible candidate kinase for REC8 phosphorylation in Arabidopsis. Therefore, I mutagenized the CDKA;1 phosphorylation sites identified in vitro and analyzed their impact on meiosis by complementation assays of the rec8 mutant phenotype. The simulations dephospho-mutation of seven sites resulted in a partial rec8 like phenotype indicating the general importance of REC8 phosphorylation. By the subsequent analysis of single mutants, I could identify one site for which a single dephospho-mutation led to aberrant meiotic procedure as well. Interestingly, rec8 mutant plants show severe defects in chromosome cohesion that leads to complete sterile plants. However, these plants are able to partially maintain centromeric cohesion almost until the end of meiosis I, leading to the question if another cohesin complex mediates sister chromatid cohesion in meiosis like it was shown for other organisms like mammals, worms and flies. Therefore, I systematically re-investigated the localization of the α-kleisins in meiosis. While RAD21.1 and RAD21.3 were only found to be expressed in vegetative cells, I found that RAD21.2, contrasting previous reports, showed a chromosomal localization during both cell divisions. Interestingly, an enrichment of RAD21.2 at heterochromatic repetitive DNA elements like ribosomal DNA (rDNA) was found. In many species, meiotic recombination in these regions is blocked by a poorly understood mechanism. The analysis of RAD21.2 RNAi knock down plants that exhibit an increase of non-homologous recombination in rDNA regions inducing genome instability, leads to the hypothesis that RAD21.2 is required for the repression of recombination at heterochromatin. Since an appearance of REC8 at rDNA regions was found in RAD21.2 RNAi knock down plants, we hypothesize that the distribution of REC8 and RAD21.2 containing cohesin complexes shape the recombination landscape in Arabidopsis meiosis.