Time-resolved reprogramming of single somatic cells into totipotent states during plant regeneration

Abstract

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.

Keywords: LEC2; SPCH; auxin biosynthesis; live imaging; single-nucleus RNA sequencing; somatic embryo founder cells; somatic embryogenesis; totipotency.