Photosynthesis-derived carbon gates cell cycle activation to enable hormone-autonomous shoot regeneration

Shoot regeneration is a powerful model for cell fate reprogramming but how it occurs in nature remains poorly understood because studies in Arabidopsis thaliana conventionally rely on in vitro assays supplemented with exogenous hormones and sugars. Here, we established the Hormone-autonomY Direct Regeneration Assay (HYDRA) in which removal of the shoot apical meristem (SAM) initiates shoot regeneration from the cotyledon-hypocotyl boundary without hormone or sugar supplementation. We identify photosynthesis-derived carbon and the boundary domain as two separable but convergent requirements for shoot regeneration in HYDRA. Following SAM removal, carbon availability increases in the boundary domain where it activates cell-cycle progression via the RETINOBLASTOMA-RELATED1 (RBR1) pathway. Carbon deprivation blocks regeneration despite induction of shoot meristem genes, indicating proliferative activation is limiting. Pre-existing shoot-competent boundary cells give rise to regenerated shoots upon cell-cycle activation, while their perturbation impairs regeneration despite sufficient carbon. Carbon therefore gates the proliferative activation of these cells, enabling functional shoot meristem formation. Additionally, exogenous carbon supply overcomes the requirement for SAM removal to induce shoot formation, indicating that carbon availability also acts as an initiation cue. Together, this study reveals an inherent capacity for hormone-autonomous shoot regeneration and identifies photosynthesis-derived carbon as a central regulator of this process.