RpFT5 Regulates Root‐Sprouting Rejuvenation in Robinia pseudoacacia Through Transcriptional Control by RpSPL9/RpSOC1 and Epigenetic Regulation via AGO4‐Mediated DNA Methylation

Root-based vegetative propagation enables mature trees to regain juvenile characteristics, a process commonly referred to as rejuvenation, yet the underlying transcriptional and epigenetic regulatory mechanisms remain poorly understood. In this study, Robinia pseudoacacia was employed as a model system owing to its strong natural sprouting capacity to dissect the molecular basis of root-derived rejuvenation. Transcriptomic profiling identified RpFT5 as a central hub gene associated with the transition between mature and juvenile states during root sprouting. Functional analyses demonstrated that RpFT5 was positively regulated by the transcription factors RpSPL9 and RpSOC1, which directly bound to distinct cis-elements in its promoter and activated its transcription. In addition, epigenetic analyses revealed that CHH DNA methylation in the RpFT5 promoter was dynamically associated with developmental phase changes and was significantly enriched in mature tissues. This methylation could be regulated through the RNA-directed DNA methylation (RdDM) pathway involving an RpAGO4-like protein. RpAGO4-like contributed to CHH methylation of the RpFT5 promoter and physically interacted with RpSPL9 and RpSOC1, attenuating their transcriptional activation of RpFT5. This coordinated transcriptional and epigenetic regulatory network could result in fine-tuned repression or activation of RpFT5 during rejuvenation, thereby modulating juvenile trait re-establishment during root sprouting. Collectively, these findings define a dual-layer regulatory mechanism integrating transcription factor activity and DNA methylation in controlling root-based vegetative rejuvenation, and propose a model in which RdDM-associated CHH methylation functions as a dynamic regulatory module governing developmental phase transitions in perennial trees.