Cell lineages of soybean anther morphogenesis reveal developmental trajectory of microsporocytes at single-cell resolution

In flowering plants, anther morphogenesis and microspore mother cell (MMC) specification of the male reproductive organ are critical for fertility and seed-setting or yield, but their underlying mechanisms in soybean (Glycine max) remain largely uncharacterized. Here, we cytologically established a staged developmental framework of early soybean anthers that were collected to generate a high-resolution single-nucleus RNA-seq atlas. We further characterized 11 major cell types annotated by cluster-specific marker genes and validated by RNA in situ hybridization. Pseudotime trajectory analysis reconstructed the differentiation process of microsporocytes, tapetum, and middle layer from archesporial cells, and established a regulatory network governing germline development. Parallel comparison of mitotic dividing cells (DCs) and meiotic MMCs revealed divergent transcriptional and cell-cycle programs. DCs follow a linear mitotic cascade with smooth, continuous transcriptional regulation, whereas MMCs undergo lineage-specific transcriptional reprogramming with an oscillatory activation of cell-cycle regulators to orchestrate meiotic transitions and progression. By comparing the transcriptional regulatory network of DCs and MMCs, we identified a pair of C2H2 zinc finger factors GmKNUa/b, the homologs of Arabidopsis KNUCKLES, as a key switch for MMC differentiation in soybean. The CRISPR-Cas9-edited Gmknua/b double mutants lack meiocytes, and GmKNUa/b might trigger the mitosis-to-meiosis transition by repressing the persistent mitotic cell cycle. Taken together, these results reveal the cellular signatures and molecular basis of early soybean anther morphogenesis and germline specialization, and provide candidate targets for manipulating germline fate and male fertility in soybean.