Single‐Cell Transcriptome Atlas and Dynamic Regulatory Mechanisms of Anther Development in Alfalfa ( Medicago sativa L.)

Anthers consist of various specialised cell types and play a significant role in plant reproduction. Although the molecular mechanisms underlying anther development and regulation have been extensively studied, the single-cell transcriptional landscape and dynamic regulation during anther development in M. sativa remain largely unexplored. In the present study, we constructed the first single-cell transcriptome atlas of M. sativa anthers to provide a comprehensive view of cell type-specific gene expression and epigenetic modifications. The reconstruction of the developmental trajectories of tapetum and microspores led to the identification of novel genes and elucidated the regulatory networks involved in tapetum formation. Our findings revealed rapid functional transitions in tapetum during the tetrad stage, including cell formation, specialisation, and programmed cell death (PCD). Additionally, we analysed the stages at which tapetal degradation and microspore shrinkage occurred in the sterile line. Overall, this study offers valuable insights into the molecular mechanisms underlying anther development in M. sativa. Specifically, MsKIN14P was identified as a key regulator of microtubules and the cytoskeleton during mitosis, and the transcription factors ERF3 and ERF025 were shown to influence anther development through the ethylene response pathways. These findings provide an essential theoretical foundation for the development of novel male-sterile lines and enhance the breeding capabilities of M. sativa.