Whole-tissue 3D immunostaining of shoot apical meristems in rice at single-cell resolution

The shoot apical meristem (SAM) produces all above-ground organs of plants and is thus a central focus of plant developmental biology. Developmental processes in the SAM are regulated by various factors that control gene expression at the cellular level. Key among these are the chemical modifications of the N-terminal tails of histones, which are essential components of nucleosomes and chromatin that play crucial roles in these processes. While immunostaining is a valuable method for the spatial analysis of histone modifications, its application to the SAM has posed technical challenges. Here, we developed a three-dimensional immunostaining method for rice (Oryza sativa) SAMs at single-cell resolution using a permeabilization process with specific cell wall degrading enzymes, along with the iTOMEI clearing technique (Sakamoto et al. [2022] Communications Biology, 5, 12). We detected clear signals throughout the deeper tissue layers, allowing us to visualize histone modifications associated with both active and repressive chromatin states, as well as M phase–specific modifications localized on chromosomes. The repressive modifications H3K9me2 and H3K27me3 exhibited punctate patterns within the nuclei, whereas the modifications linked to transcriptional activity were more diffusely distributed. Double staining showed that H3K9me2 forms a peripheral layer around a central domain enriched in H3K4me1. A comparative analysis of SAMs during the vegetative and reproductive phases indicated that active modifications persisted across both phases, whereas repressive modifications increased during the reproductive phase. Our protocol facilitates the three-dimensional visualization of chromatin states in the SAM, offering a robust tool for exploring the spatial regulation of plant development at the single-cell level.