GmCEP1 affects soybean hard seededness via palisade cells and chemical composition

Hard seededness, a crucial trait in soybean [Glycine max (L.) Merr.] domestication, which can enhance survival in harsh environments. However, it also has a negatively impact yield and processing quality. Paraffin section analysis revealed a significant difference in seed coat thickness between the hard-seeded mutant m_zp661 and wild-type seeds at the R8 stage, particularly in the palisade tissue layer. The hard characteristic of m_zp661 was not related to the ABA and GA content in the seeds. However, a higher calcium ion content and a lower pectin content were measured in m_zp661, which may be the cause of reduced seed coat permeability. To analyze its molecular mechanism, BSA-seq was utilized to examine the recombinant inbred lines (RILs) with differences in seed imbibition constructed from m_zp661 and Zhonghuang 13. Based on the gene annotation analysis and gene expression pattern analysis, the GmCEP1, specifically expressed in seed coat and associated with hard seed traits, was screened. In situ hybridization revealed a higher expression of GmCEP1 in the palisade cells of the seed coat. Repressed seed germination, reduced seed size, and increased seed coat thickness were observed in GmCEP1 overexpressing lines. Furthermore, the transcriptional activity of pGmCEP1^H-II was found to be significantly higher than that of pGmCEP1^H-I, indicating that natural variation could influence seed hardening by modulating the transcription of GmCEP1. Overall, this research enhances our understanding of the molecular mechanisms underlying soybean hard seededness, providing insights for the development of soybean varieties with high germination rates.