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

## TL;DR

This study identifies a genetic pathway in apple plants that helps form embryogenic cells during early somatic embryogenesis by breaking down cell walls.

## Contribution

The novel contribution is the discovery of the eTM–miR3699–MAN7 regulatory module's role in cell wall degradation and embryogenic cell formation in apple.

## Key findings

- MdMAN7 overexpression enhances β-mannanase activity and promotes hemicellulose degradation.
- miR3699 negatively regulates MdMAN7, while eTM3699 acts as a target mimic to prevent MdMAN7 cleavage.
- The eTM–miR3699–MAN7 module positively regulates embryogenic cell formation and improves somatic embryogenesis efficiency.

## Abstract

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 MANNAN7 (MAN7) 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–MdMAN7. The overexpression and CRISPR/Cas9-mediated transgenic assays were used for functional analysis of miR3699 and MdMAN7. MdMAN7 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 MdMAN7 can significantly improve the efficiency and shorten the induction cycle of SE. miR3699 acted by negatively regulating MdMAN7. In addition, eTM3699 were identified as endogenous target mimics of miR3699 that bind to miR3699 to prevent cleavage of MdMAN7 and thereby positively regulate embryogenic cell formation. In conclusion, our results elucidate the mechanism of eTM–miR3699–MAN7 module regulating embryogenic cell formation during the early stage of SE in apple.