Auxin signaling mediated embryogenic cell formation during the early stage of somatic embryogenesis in Malus domestica “Gala”

Embryogenic cell formation is a prerequisite for somatic embryogenesis (SE) in plants. Auxin signaling is indispensable for the initiation of embryogenic cell formation. However, the mechanism of auxin signaling that regulates this process in apple remains unclear. In this study, the MdAUX/IAA7.1 and MdARF5 genes were identified as key auxin signaling regulators in the early stage of SE through RNA sequencing and RT-qPCR in Malus domestica "Gala." MdARF5 significantly promoted embryogenic cell formation and shortened the induction cycle of SE in transgenic overexpression assays. When MdARF5 was knocked out, the explants could not form somatic embryos even when stimulated with exogenous auxin. Through RNA sequencing analysis of MdARF5-overexpressing lines and transgenic assays, MdLBD41 was identified as the key gene that regulates SE and responds to MdARF5. In response to auxin signaling, the MdARF5 protein directly binds to the promoter of MdLBD41 to activate its transcription, thereby promoting SE in apples. MdAUX/IAA7.1 interacts with the MdARF5 protein without auxin signaling, inhibiting the downstream transcription of MdLBD41, and thereby negatively regulating SE in apples. In conclusion, our results elucidate the mechanism of MdAUX/IAA7.1 and MdARF5 genes responding to auxin and MdARF5, promoting the embryogenic cell formation by mediating the positive regulation of MdLBD41 expression.