PtWOX2 and PtWOX14 accelerate somatic embryogenesis initiation in Pinus massoniana via synergistic hormonal and transcriptional reconfiguration

Abstract

Somatic embryogenesis (SE) represents a powerful tool for conifer biotechnology, yet its application in Pinus massoniana is frequently hindered by severe genotype-dependent recalcitrance. We identified two WUSCHEL-related homeobox ( WOX ) orthologs, PtWOX2 and PtWOX14 , as key regulators driving the embryogenic transition. By employing a genetic transformation system harboring the RUBY reporter for real-time visual selection, we demonstrated that ectopic expression of PtWOX2 and PtWOX14 dramatically accelerated SE initiation, with proembryogenic masses (PEMs) emerging 22.0-26.7 days earlier than in controls. Notably, PtWOX overexpression enabled the “fate reversal” of recalcitrant cell lines, transforming disorganized cell aggregates into highly polarized PEMs with distinct embryonal heads. Maturation capacity was increased by up to 1.7-fold in PtWOX2 -overexpressing lines, whereas PtWOX14 exhibited a genotype-dependent effect on somatic embryo yield. Physiological and WGCNA analyses revealed that PtWOX genes orchestrate a reciprocal hormonal reconfiguration, shifting the hormonal landscape from a high-auxin proliferative state to a low-IAA/high-ABA differentiation-permissive state. This transition is sustained by hierarchical regulatory networks in which PtWOX2 and PtWOX14 converge on an IAA-mediated execution module. This process involves the mobilization of master hubs such as bHLH139 and HB9 , to activate cell-wall and metabolic enzymes ( MAN1 , Cht1 , and EXPA1 ) while relieving the transcriptional repression imposed by auxin signaling inhibitors like IAA11 . Furthermore, protein interaction assays confirmed that PtWOX2 associates with nuclear partners such as Pt1G46750 and Pt5G39360, likely forming a post-translational regulatory complex to coordinate downstream developmental programs. Our study demonstrates that PtWOX2 and PtWOX14 significantly accelerate the initiation of somatic embryos in P. massoniana , offering a comprehensive molecular framework to address conifer recalcitrance via PtWOX -mediated synergistic hormonal and transcriptional reprogramming. Furthermore, by establishing a genetic transformation system integrated with the RUBY reporter, this work provides a potential foundation for the genetic improvement and future large-scale propagation of Pinus species by enhancing early developmental efficiency.