Identification of miRNA-mRNA regulatory network during the germination of soybean seed (Glycine max) and the role of Gma-miR1512a-GmKIN10 interaction

Seed germination is a key and complex physiological process in plant life, including soybeans. Here, we explored the miRNA-mRNA transcriptome changes and the key genes in the germination stages of the soybean. Morphological analysis showed that the imbibition of seeds was completed at 12 h, and the embryo broke through the seed coat at 36 h. During seed germination, mRNA and miRNA sequencing identified 20845 differentially expressed mRNAs (DEMs) and 421 differentially expressed miRNAs (DEMIs) at three specific time points: 12 h, 36 h, and 108 h. KEGG enrichment revealed that plant hormone signal transduction, plant-pathogen interaction and MAPK signaling pathway-plant were the crucial biological processes for seed germination. ABA and GA related DEMs on plant hormone signal transduction were abundant. miRNA-mRNA integrated analysis showed that 5170 miRNA-mRNA pairs were found. During germination, 20 significant miRNA-mRNA interactions were identified, involving the top 10 differentially expressed miRNAs (DEMIs) and 198 differentially expressed mRNAs (DEMs). Interestingly, the expression level of Gma-miR1512a increased significantly during soybean seed germination. This miRNA specifically regulates GmKIN10, homologous to AtKIN10, which mediates germination. To verify this interaction, co-agroinjection of GmKIN10-GFP/GUS and Gma-miR1512a into tobacco leaves demonstrated that Gma-miR1512a can inhibit GmKIN10 expression by cleaving its target site. Furthermore, the function of Gma-miR1512a-GmKIN10 were verified by overexpression transgene. Although Arabidopsis seeds overexpressing Gma-miR1512a (OE-Gma-miR1512a) showed no significant differences in germination indices compared to wild-type (WT) seeds, those overexpressing GmKIN10 (OE-GmKIN10) exhibited significantly lower germination indices. The seeds germination index of GmKIN10 and Gma-miR1512a double overexpression lines recovered. Additionally, the yeast two-hybrid assay, protein interaction prediction,and molecular docking all showed that GmKIN10 might interact with GmPP2A and GmDSP4. This study identified a complex miRNA-mRNA regulatory network that plays a crucial role in soybean seed germination. Specifically, Gma-miR1512a was found to regulate GmKIN10, significantly influencing germination rates and hormone signaling pathways.