CitRWP directly activates CsDAZ3 to promote somatic embryogenesis in Citrus

Somatic embryogenesis (SE) serves as an ideal model for studying plant cell totipotency and embryonic development mechanisms, yet the transcriptional and epigenetic regulation underlying this process remains poorly characterized. We found that citrus nucellar polyembryony (a form of apomixis) gene, RWP-RK domain-containing transcription factor (CitRWP) is highly expressed in embryogenic callus with strong SE capacity, and its expression is constantly upregulated during SE induction. Overexpression of CitRWP induced spontaneous somatic embryo formation in a citrus variety that is resistant to SE bypass exogenous inducer. Suppression of CitRWP expression by RNA interference (RNAi) impairs SE capacity. Integrated RNA-seq and Chromatin immunoprecipitation sequencing (ChIP-seq) analyses combined with molecular assays revealed that CitRWP directly binds to the DUO1-ACTIVATED ZINC FINGER 3 (CsDAZ3) promoter and activates its transcription. Overexpression of CsDAZ3 significantly enhanced SE capacity. Comparative analysis of ChIP-seq and ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) datasets demonstrated that CitRWP is capable of binding to regions of low chromatin accessibility. CitRWP binds to a condensed chromatin site in the CsDAZ3 promoter and promotes increased local chromatin accessibility at the CsDAZ3 locus. Our findings delineate a novel regulatory framework for citrus SE, highlighting that CitRWP coordinates transcriptional regulation with chromatin remodeling. This research provides insights into plant cell reprogramming and provides pivotal molecular targets for optimizing breeding protocols by modulating SE.