The extracellular regulatory network and key factors governing early embryogenesis in Nicotiana tabacum

The plant extracellular space, including the cell wall, is a dynamic signaling compartment outside the plasma membrane that plays crucial roles in cell-cell communication and developmental coordination. Although early embryogenesis is known to be orchestrated by both intracellular and extracellular cues, the molecular mechanisms underlying extracellular regulation remain largely unexplored. Here, we report that NtProRP1, an extracellular protein, localizes to the cell wall immediately after fertilization in Nicotiana tabacum. Targeted knockout of NtProRP1 using the CRISPR-Cas9 system resulted in arrested embryos with irregular cell shapes and aberrant division patterns in aborted seeds. Furthermore, we performed high-resolution transcriptome profiling on a limited number of isolated two-celled proembryos from wild-type (WT) and Ntprorp1 plants, enabling the detection of early transcriptional changes with minimal interference from surrounding tissues. Genes involved in the synthesis and modification of cell wall components, including cellulose, hemicellulose, and pectin, were coordinately upregulated at the onset of embryogenesis in the Ntprorp1 mutant. Further analysis suggests a possible role for NtProRP1 in cell wall formation via the cell wall integrity (CWI) maintenance mechanism, potentially through the involvement of components such as receptor-like kinases (RLKs), mitogen-activated protein kinases (MAPKs), Ca2+ flux, and multiple phytohormones. Together, these results uncover a new mechanism for NtProRP1 in regulating early embryogenesis and deliver a unique transcriptomic resource that advances understanding of extracellular signaling in plant embryogenesis.