The CitRWP–CsWUS regulatory axis governs citrus plant regeneration in association with chromatin remodeling

Plant regeneration underpins crop genetic improvement, yet its regulatory mechanism remains poorly characterized in woody fruit trees. The RWP-RK domain-containing (RKD) transcription factor CitRWP acts as a pleiotropic regulator of nucellar polyembryony and somatic embryogenesis (SE) in citrus, but its downstream molecular network remains elusive. We demonstrate that CsWUS, a core stem cell regulator, is a direct downstream target of CitRWP. CitRWP binds to the CsWUS promoter to activate its transcription. Additionally, CitRWP interacts with the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin-remodeling ATPase SPLAYED (SYD), synergistically enhancing CsWUS activation, while chromatin accessibility at the CsWUS locus is enhanced upon CitRWP overexpression. We further uncover that CsWUS negatively regulates CitRWP expression via a feedback loop. CsWUS overexpression promotes SE in recalcitrant citrus callus, but causes abnormal meristem proliferation and impairs shoot organogenesis from epicotyl segments. CsWUS greatly enhanced in planta transformation efficiency in the model citrus. This study establishes the CitRWP-CsWUS regulatory axis as a central module that coordinates multiple developmental fate transitions in association with chromatin remodeling, providing novel insights into the mechanisms of cellular totipotency and plant regeneration.