Multiscale chromatin dynamics and high entropy in plant iPSC ancestors

Plant protoplasts provide a starting material to induce pluripotent cell masses in vitro competent for tissue regeneration. Dedifferentiation is associated with large-scale chromatin reorganisation and massive transcriptome reprogramming, characterized by stochastic gene expression. How this cellular variability reflects on chromatin organisation in individual cells and what are the factors influencing chromatin transitions during culturing is largely unknown. High-throughput imaging and a custom, supervised image analysis protocol extracting over 100 chromatin features unravelled a rapid, multiscale dynamics of chromatin patterns which trajectory strongly depends on nutrients availability. Decreased abundance in H1 (linker histones) is hallmark of chromatin transitions. We measured a high heterogeneity of chromatin patterns indicating an intrinsic entropy as hallmark of the initial cultures. We further measured an entropy decline over time, and an antagonistic influence by external and intrinsic factors, such as phytohormones and epigenetic modifiers, respectively. Collectively, our study benchmarks an approach to understand the variability and evolution of chromatin patterns underlying plant cell reprogramming in vitro.

Methods
Image features extracted by Tissue Maps from microscopy images following automated, supervised segmentation of plant protoplast nuclei. 

Leaf protoplasts  were cultured in coverglass-bottom 96-well plates and imaged using a confocal microscope Cell Voyager. The plant cell line (expressing  specific nuclear reporters), imaging day (day0-day7) and culturing media is indicated in the dataset. Additional information is provided in the Table S3 in the manuscript.

Image features correspond to three families: intensity, morphology and texture. A complete list of image features used for the study is available as Table S2 in the manuscript.