Effects of hybridization and polyploidy on the histone H3 phosphorylation at serine 10 (H3S10ph) in 'Pennisetum' spp. Rich. (Poaceae)

Abstract

Post-translational modifications on N-terminal tails of histones form nucleosomes, which are often associated with distinct biological functions. Some theories suggest that one of these changes, the phosphorylation of histone H3 at serine 10 (H3S10ph) plays an important role in chromosome condensation and sister chromatid cohesion. Although the histones are highly conserved, studies have been shown that the role and distribution of H3S10ph may differ between species. This study evaluated the effects of interspecific hybridization and polyploidization on the histone H3 phosphorylation at serine 10 (H3S10ph), using two model species 'Pennisetum purpureum' Schum (elephant grass; 2n=4x=28) and 'Pennisetum glaucum' (L.) R. Br. (Millet; 2n=2x=14). These species differ in ploidy levels and their hybrids are sterile. The fertility is restored by inducing chromosome doubling with colchicine. The effectiveness of inducing polyploidy and genomic behavior of duplicated hybrids were verified by meiotic analysis, chromosome counting in mitotic metaphase, cell cycle analysis and estimates of DNA content by flow cytometry. The H3S10 phosphorylation pattern was evaluated by immune-detection technique during the cell cycle in 'Pennisetum purpureum', 'Pennisetum glaucum' and their triploid and hexaploid hybrids. First, roots were fixed in 4% paraformaldehyde. We primarily applied antibody (rabbit polyclonal IgG), which was detected with a secondary antibody (goat anti-rabbit IgG-FITC). The pattern of H3S10ph during mitosis showed coordination in space and time. The pattern was similar in parents and hybrids chromosomes. Phosphorylation was coincident with cohesion restricted to pericentromeric region. In triploid and partial hexaploid hybrids, bridges in anaphase and telophase were found, which showed signals of phosphorylation of H3S10, suggesting loss of chromosome fragments, mainly due the process of hybridization and polyploidization. In the hybrid hexaploid, metaphases with non-oriented chromosomes and anaphases with delayed and lost chromosomes with and without immune signal were found. This suggests that, after polyploidization, even chromosomes with functional centromeres, identified by phosphorylation of H3S10ph, are eliminated in hybrid 'Pennisetum', indicating that inactivation of the centromere is not a factor that contributes to chromosome elimination in 'Pennisetum' partial hexaploid hybrids.