The acquisition of pluripotent callus from somatic cells plays an important role in plant development studies and crop genetic improvement. This developmental process incorporates a series of cell fate transitions and reprogramming. However, our understanding of cell heterogeneity and mechanisms of cell fate transition during callus induction remains quite limited. Here, we report a time-series single-cell transcriptome experiment on Arabidopsis root explants that were induced in callus induction medium for 0, 1, and 4 days, and the construction of a detailed single-cell transcriptional atlas of the callus induction process. We identify the cell types responsible for initiating the early callus: lateral root primordium-initiating (LRPI)-like cells and quiescent center (QC)-like cells. LRPI-like cells are derived from xylem pole pericycle cells and are similar to lateral root primordia. We delineate the developmental trajectory of the dedifferentiation of LRPI-like cells into QC-like cells. QC-like cells are undifferentiated pluripotent acquired cells that appear in the early stages of callus formation and play a critical role in later callus development and organ regeneration. We also identify the transcription factors that regulate QC-like cells and the gene expression signatures that are related to cell fate decisions. Overall, our cell-lineage transcriptome atlas for callus induction provides a distinct perspective on cell fate transitions during callus formation, significantly improving our understanding of callus formation.

Totipotency enables single cells to regenerate an organism, yet how differentiated somatic cells reacquire this potential remains unclear. Here, we show that LEAFY COTYLEDON2 (LEC2) reprograms SPEECHLESS (SPCH)-expressing meristemoid mother cells (MMCs) away from stomatal-lineage progression, driving their conversion into totipotent somatic embryo founder cells (SEFCs) in Arabidopsis cotyledons. Using time-course live imaging, single-nucleus RNA sequencing (snRNA-seq), and spatial laser capture microdissection combined with RNA sequencing (LCM-RNA-seq), we uncover a lineage bifurcation point where MMC derivatives either commit to guard cells or transition into a guard mother cell (GMC)-auxin intermediate, an auxin-enriched state that enables transcriptional reprogramming and embryonic gene activation. LEC2 and SPCH cooperatively activate TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) and YUCCA4 (YUC4), establishing a local auxin biosynthesis circuit essential for SEFC specification. Genetic and promoter analyses confirm MMCs as the origin of somatic embryos, with TAA1/YUC-mediated auxin production indispensable for totipotency and embryogenesis. These findings define an auxin-driven, transcriptionally regulated trajectory linking stomatal progenitors to somatic embryogenesis, revealing a direct route that advances mechanistic understanding of plant regenerative plasticity.

Summary: Understanding the complex functions of plant leaves requires spatially resolved gene expression profiling with single-cell resolution. However, although in situ gene expression profiling technologies have been developed, this goal has not yet been achieved. Here, 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. We successfully characterized subtle but significant transcriptomic differences between upper and lower epidermal cells. Furthermore, with high-resolution location information, we discovered the cell type-specific spatial gene expression gradients from main vein to leaf edge. By reconstructing those spatial gradients, we show for the first time the distinct spatial developmental trajectories of vascular cells and guard cells. Our findings show the importance of incorporating spatial information for answering complex biological questions in plant, and scStereo-seq offers a powerful single cell spatially resolved transcriptomic strategy for plant biology.

De novo shoot regeneration from a callus plays a crucial role in both plant biotechnology and the fundamental research of plant cell totipotency. Recent studies have revealed many regulatory factors involved in this developmental process. However. our knowledge of the cell heterogeneity and cell fate transition during de novo shoot regeneration is still limited. Here, we performed time-series single-cell transcriptome experiments to reveal the cell heterogeneity and redifferentiation trajectories during the early stage of de novo shoot regeneration. Based on the single-cell transcriptome data of 35,669 cells at five-time points, we successfully determined seven major cell populations in this developmental process and reconstructed the redifferentiation trajectories. We found that all cell populations resembled root identities and undergone gradual cell-fate transitions. In detail, the totipotent callus cells differentiated into pluripotent QC-like cells and then gradually developed into less differentiated cells that have multiple root-like cell identities, such as pericycle-like cells. According to the reconstructed redifferentiation trajectories, we discovered that the canonical regeneration-related genes were dynamically expressed at certain stages of the redifferentiation process. Moreover, we also explored potential transcription factors and regulatory networks that might be involved in this process. The transcription factors detected at the initial stage, QC-like cells, and the end stage provided a valuable resource for future functional verifications. Overall, this dataset offers a unique glimpse into the early stages of de novo shoot regeneration, providing a foundation for a comprehensive analysis of the mechanism of de novo shoot regeneration.

Callus is a reprogrammed transitional cell mass during plant regeneration. Pluripotent callus cells develop into fertile shoots through de novo shoot organogenesis. This study represents a pioneering effort in exploring the spatial transcriptome of tomato callus during shoot regeneration, using technologies including BGI Stereo-seq, BMKMANU S1000, and 10x Visium. The results indicate that the callus comprises highly heterogeneous cells, classified into various cell types based on spatial gene expression and histological observation, including epidermis, shoot primordium, vascular tissue, inner callus, and outgrowth shoots. The developmental trajectories from shoot primordium to outgrowth shoot are traced, and vascular tissue development is characterized. The single-cell resolution spatial approach reveals the origin of shoot primordia from the sub-epidermis. The spatial full length RNA sequencing shows high incompletely spliced (IS) ratios in the shoot primordium cells. These findings enhance our knowledge of plant organogenesis and highlight the significance of spatial biology in plant research.