We present the first in situ single-cell transcriptome profiling in plant, scStereo-seq (single-cell SpaTial Enhanced REsolution Omics-sequencing), which enabled the bona fide single-cell spatial transcriptome of Arabidopsis leaves.

Transitions between root and shoot identities are central to plant development and regeneration. Here, we identify a transitional cell (TC) state in Arabidopsis thaliana that bridges these identity switches during de novo shoot regeneration, with parallels suggested during embryogenesis. This TC state consists of two distinct populations: a shoot progenitor population and surrounding boundary cells that collaborate to form a functional shoot system. During a brief transitional phase, these cells exhibit overlapping root- and shoot-specific gene expression, orchestrated by antagonistic interactions between the homeotic factors ATML1 and WOX5. MYB31 is identified as a key regulator that shifts this balance toward shoot formation. By integrating single-nucleus RNA sequencing and spatial transcriptomics, we delineate the molecular signatures, spatial organization, and developmental trajectories of these populations. This study provides a conceptual framework for understanding root-to-shoot transitions and lays the groundwork for engineering plant regeneration, with applications in crop improvement and sustainable agriculture.