Super-resolution multimodal spatial transcriptomics reveals an ovoid stem cell niche structuring de novo shoot regeneration.

Plant de novo regeneration, unlike regeneration in most animal systems, relies on callus formation and re-establishment of stem cell niches (SCNs) rather than on pre-existing reservoirs. Growth hormones such as auxin and cytokinin, together with numerous genetic regulators, participate in regeneration, but the architectural principles coordinating extensive cellular reprogramming at the individual-cell scale remain unknown. In this sduty, we used super-resolution multimodal spatial transcriptomics to profile approximately 1.2 million cells during tomato shoot regeneration from wounding to organogenesis. This spatiotemporal atlas resolved vascular-associated callus initiation, epidermal and pluripotent cell-state transitions, and the organization of Wuschel-expressing cells during niche formation. High-resolution spatial imaging identified a previously unrecognized ovoid-structured SCN composed of a peripheral signaling layer, an intermediate plastic compartment, and a central quiescent core. Within this architecture, EPFL8b is enriched in the signaling layer, whereas its receptors, ER and ERL1, are mainly localized to the plastic compartment. Genetic, peptide treatment, protein interaction, and spatial transcriptomic analyses showed that EPFL8b-mediated signaling contributes to SCN organization and shoot regeneration. Together, these findings expand traditional gene-centric paradigms, highlighting structural organization as a fundamental principle in regeneration and providing a high-resolution framework for studying SCN formation.