Single-cell RNA sequencing reveals developmental trajectories and environmental regulation of callus formation in Arabidopsis.

Plant cells exhibit an extraordinary regenerative potential, achieving cellular totipotency by dedifferentiating to form new tissues. While significant progress has been made in understanding cell fate mechanisms, the regulatory networks governing callus cell development remain insufficiently explored, particularly regarding cell classification, morphology, and regulatory processes. This study provides a detailed investigation into the developmental dynamics and transcriptomic profiles of callus cells in Arabidopsis at key stages: initiation, proliferation, and greening. Employing single-cell RNA sequencing and UMAP-based clustering, we annotated cell clusters based on highly enriched gene expressions. Developmental trajectories were further mapped through pseudotime analysis, revealing distinct transcription factor networks. Additionally, functional analysis of key regulatory genes was conducted using mutant and overexpression lines, affirming their roles in callus development. Gene Ontology analysis highlighted the involvement of environmental factors-low oxygen and salinity promoted callus formation, while light inhibited it, though essential for greening. These findings shed light on the complex regulatory landscape of plant tissue regeneration and guide future research avenues.