Cryptic Fate Priming and Lineage Stabilization during Arabidopsis Anther Development

Plant germ cells are specified de novo from somatic tissues, yet how early anther progenitors initiate divergent germline and somatic fates remains unclear. Here, we generated a high-resolution single-cell transcriptomic atlas of Arabidopsis anther development, profiling 66,864 cells from early lineage specification through pollen maturation. We resolved major anther cell types and identified an early L2-derived intermediate population, termed the archesporial-derived state (Ar-d), marked by REM22, ER/ERL1/2, SPL/NZZ, and BAM1. Although classical anatomical studies define archesporial cells, primary sporogenous cells, primary parietal cells, and secondary parietal derivatives based on developmental position and inferred lineage relationships, our analysis showed that these early L2-derived intermediates remain globally similar, forming a shared progenitor-like transcriptional state before lineage stabilization. Trajectory reconstruction, RNA velocity, and module analyses revealed that germline and somatic trajectories first emerge through restricted branch-biased transcriptional programs, which are later amplified into robust lineage-specific identities. Mutant single-cell analyses defined two regulatory steps: SPL/NZZ establishes early branch-associated transcriptional programs within the Ar-d state, whereas EMS1 stabilizes the somatic/tapetal trajectory and restricts inappropriate enrichment of germline-associated states. Lineage-resolved analyses further defined maturation programs in germline, tapetum, middle layer, and endothecium cells, and identified LBD transcription factors required for normal pollen development. Together, our study reveals a cryptic mode of anther fate priming in which shared early L2-derived intermediates progressively transform limited branch-biased transcriptional programs into distinct reproductive and somatic cell identities through SPL-dependent priming and EMS1-dependent somatic/tapetal stabilization.