Knockdown of CENH3 in Arabidopsis reduces mitotic divisions and causes sterility by disturbed meiotic chromosome segregation.

The histone H3 variant (CENH3) of centromeric nucleosomes is essential for kinetochore assembly and thus for chromosome segregation in eukaryotes. The mechanism(s) that determine centromere identity, assembly and maintenance of kinetochores are still poorly understood. Although the role of CENH3 during mitosis has been studied in several organisms, little is known about its meiotic function. We show that RNAi-mediated CENH3 knockdown in Arabidopsis thaliana caused dwarfism as the result of a reduced number of mitotic divisions. The remaining mitotic divisions appeared to be error-free. CENH3 RNAi transformants had reduced fertility because of frequently disturbed meiotic chromosome segregation. N-terminally truncated EYFP-CENH3(C) is deposited to and functional within Arabidopsis centromeres of mitotic chromosomes, but cannot be loaded onto centromeres of meiotic nuclei. Thus the N-terminal part is apparently required for CENH3 loading during meiosis. EYFP-CENH3(C) expression reduces the amount of endogenous CENH3, thus mimicking the effect of RNAi. The consequences of reduced endogenous CENH3 and lack of meiotic incorporation of EYFP-CENH3(C) are reduced fertility caused by insufficient CENH3 loading to the centromeres of meiotic chromosomes, subsequent lagging of chromosomes and formation of micronuclei.