FANS uncovers WOX2-associated early regulators of auxin-induced somatic embryogenesis in Arabidopsis

Somatic embryogenesis (SE), the process by which differentiated somatic cells are reprogrammed to form embryos, represents a unique manifestation of plant cell totipotency. Despite its fundamental and applied importance, the molecular mechanisms that initiate embryogenic reprogramming remain largely unknown, mainly because explant tissues are cell-type heterogeneous and contain only a small fraction of SE-competent cells. Here, we applied fluorescence-activated nuclei sorting (FANS) in Arabidopsis thaliana to isolate cells expressing the WOX2 gene, a marker for early embryogenesis. Comparative transcriptomic analysis of WOX2⁽⁺⁾-positive and WOX2^(-)-negative nuclei revealed that genes up-regulated in the embryogenic nuclei were strongly enriched in biological processes related to embryo and tissue development, while down-regulated transcripts were linked to primary metabolism, suggesting a transcriptional switch from differentiated to totipotent identity. Several transcription factor genes showed strong induction, including MYB46, ZAT14, MYB98, GRF7, MYR2, and TCP19. Functional analyses using β-estradiol-inducible overexpression and loss-of-function mutants confirmed that these genes modulate embryogenic competence. In particular, MYB46 and ZAT14 emerged as candidate regulators acting downstream of WOX2 to coordinate complementary aspects of cell wall remodeling required for cellular isolation and the acquisition of embryogenic identity. These findings reveal an early, WOX2-centered regulatory network that precedes the activation of canonical SE regulators such as LEC2 and BBM, uncovering a new transcriptional layer of totipotency control.