Semi-automated quantification of phosphorylation signals of a histone involved in cell division in Arabidopsis

Cell division is regulated by numerous post-translational modifications of histones. Among these, phosphorylation of histone H3 at serine 10 is particularly critical for ensuring accurate chromosome segregation during mitosis. This study aimed to precisely characterise the dynamics of this phosphorylation during mitosis in plant cells, in order to better understand the underlying molecular pathways.

To achieve this, a 3D image analysis pipeline was developed based on the immunolocalisation of H3S10ph, enabling semi-automated quantification of H3S10 phosphorylation in mitotic cells of Arabidopsis roots. Additionally, a novel method was established to correct for signal attenuation with image depth, a common limitation of 3D imaging. This approach relies on directly measuring the intensity of objects of interest and quantifying their attenuation through the image stack. By accounting for the signal attenuation profile, this correction significantly enhances both the precision and statistical robustness of the analyses. Using this pipeline, subtle differences in H3S10ph levels were observed in mutants with altered PP2A phosphatase activity, suggesting an indirect role of PP2A enzymes in regulating H3S10ph levels.

This tool provides new opportunities to study these regulatory pathways in plants, taking advantage of the extensive genetic resources available in Arabidopsis. It can also be adapted to investigate other post-translational histone modifications or, more generally, any discrete 3D signal.”


Research developed at the Institute Jean-Pierre Bourgin for Plant Sciences in collaboration.