Characterization of Arabidopsis Mutants with Minimal Level of Centromeric Histone: Impact on Chromosome Segregation and Induction of Haploid Plants

Understanding fundamental principles of chromosome segregation machinery offers new opportunities for plant genome manipulation and engineering. The centromere-specific histone 3, CENH3, is an essential component of the centromere that determines assembly of functional kinetochores and hence, segregation and genetic transmissivity of chromosomes. Recently, a new method for induction of haploid plants based on modification of the centromeric histone has been described. This mechanism, called centromere-mediated genome elimination, is characterized by complete loss of a parental genome marked with a modified centromeric histone. This new method for plant haploidization can be used for rapid construction of recombinant inbred lines or generating complex mutants.

In this study we have isolated a novel allele (cenh3-2) of the Arabidopsis thaliana CENH3 gene that causes 100 fold reduction in the protein level. The cenh3-2 allele was found in a forward genetic screen aimed to identify mutants that can rescue infertility of plants deficient in proper completion of meiosis. This mutation leads to alternative splicing resulting in very low levels of functional transcript. Surprisingly, despite the drastic reduction of CENH3 in the centromeres, affected chromosomes are mitotically stable and exhibit only minor defects in meiotic segregation. This indicates that the functional centromere in plants may span a much smaller region than it has been postulated, facilitating the design of more efficient artificial chromosomes. Interestingly, cenh3-2 plants are very inefficient in haploid induction, indicating that the mechanism of genome elimination is not associated with low levels of CENH3 (weak centromere) but instead with alteration of CENH3 protein structure.

Our results are important for addressing the concept of the minimal centromere in plants and for understanding the mechanism of haploid induction.