The eTM–miR3699–MAN7 mediated cell wall degradation in regulating embryogenic cell formation during the early stage of somatic embryogenesis in apple

Somatic embryogenesis (SE) in plants requires the prior formation of embryogenic cells in plants. The remodeling of the cell wall in mature somatic cells is a prerequisite for embryogenic cell formation. However, the mechanism of this process remains unelucidated. In this study, eTM3699, miR3699, and <i>MANNAN7</i> (<i>MAN7</i>) were identified as key regulators of embryogenic cell formation through whole-transcriptome sequencing. The dual-luciferase reporter assays and GUS histochemical staining assays, were used to identified the regulatory network of eTM3699-miR3699-<i>MdMAN7</i>. The overexpression and CRISPR/Cas9-mediated transgenic assays were used for functional analysis of miR3699 and <i>MdMAN7</i>. <i>MdMAN7</i> overexpression can enhance the activity of β-mannanase, induce hemicellulose degradation, reshape the cell wall of highly differentiated somatic cells, and relieve the restriction on cell differentiation and division, ultimately positively regulating the embryogenic cell formation. Specifically, the overexpression of <i>MdMAN7</i> can significantly improve the efficiency and shorten the induction cycle of SE. miR3699 acted by negatively regulating <i>MdMAN7.</i> In addition, eTM3699 were identified as endogenous target mimics of miR3699 that bind to miR3699 to prevent cleavage of <i>MdMAN7</i> and thereby positively regulate embryogenic cell formation. In conclusion, our results elucidate the mechanism of eTM-miR3699-<i>MAN7</i> module regulating embryogenic cell formation during the early stage of SE in apple.