GhRCD1 regulates cotton somatic embryogenesis by modulating the GhMYC3-GhMYB44-GhLBD18 transcriptional cascade.

Plant somatic embryogenesis (SE) is a multifactorial developmental process where embryos that can develop into whole plants are produced from somatic cells rather than through the fusion of gametes. The molecular regulation of plant SE, which involves the fate transition of somatic cells into embryogenic cells, is intriguing yet remains elusive. We deciphered the molecular mechanisms by which GhRCD1 interacts with GhMYC3 to regulate cell fate transitions during SE in cotton. While silencing of GhMYC3 had no discernible effect on SE, its overexpression accelerated callus formation, and proliferation. We identified two of GhMYC3 downstream SE regulators, GhMYB44 and GhLBD18. GhMYB44 overexpression was unconducive to callus growth but bolstered EC differentiation. However, GhLBD18 can be triggered by GhMYC3 but inhibited by GhMYB44, which positively regulates callus growth. On top of the regulatory cascade, GhRCD1 antagonistically interacts with GhMYC3 to inhibit the transcriptional function of GhMYC3 on GhMYB44 and GhLBD18, whereby a CRISPR-mediated rcd1 mutation expedites cell fate transition, resembling the effects of GhMYC3 overexpression. Furthermore, we showed that reactive oxygen species (ROS) are involved in SE regulation. Our findings elucidated that SE homeostasis is maintained by the tetrapartite module, GhRCD1-GhMYC3-GhMYB44-GhLBD18, which acts to modulate intracellular ROS in a temporal manner.