A Two-Step Model for de Novo Activation of <i>WUSCHEL</i> during Plant Shoot Regeneration

Abstract

Plant cells are totipotent and competent to regenerate from differentiated organs. It has been known for six decades that cytokinin-rich medium induces shoot regeneration from callus cells. However, the underlying molecular mechanism remains elusive. The homeodomain transcription factor WUSCHEL (WUS) is essential for de novo establishment of the shoot stem cell niche in <i>Arabidopsis thaliana</i> We found that WUS-positive (WUS⁺) cells mark the shoot progenitor region during regeneration. A cytokinin-rich environment initially promotes the removal of the repressive histone mark H3K27me3 at the <i>WUS</i> locus in a cell cycle-dependent manner. Subsequently, the B-type ARABIDOPSIS RESPONSE REGULATORs (ARRs) ARR1, ARR2, ARR10, and ARR12, which function as transcriptional activators in the cytokinin signaling pathway, spatially activate <i>WUS</i> expression through binding with microRNA165/6-targeted HD-ZIP III transcription factors. Thus, our results provide important insights into the molecular framework for cytokinin-directed shoot regeneration and reveal a two-step mechanism for de novo activation of <i>WUS</i>.