60Co-γ irradiation induces dumbbell-shaped leaf polarity abnormalities by inhibiting PeAS1 and PeKAN2 in Phalaenopsis equestris

The establishment of adaxial-abaxial polarity in leaves is fundamental to their planar growth, regulated by intricate genetic networks. While ⁶⁰Co-γirradiation is a widely used physical mutagen for generating novel plant germplasm, the molecular links between radiation-induced morphological variations and endogenous developmental programs remain unclear. Using Phalaenopsis equestris as a model, we observed that 40Gy 60Co-γirradiation significantly induces dumbbell-shaped heteromorphic leaf phenotypes. Morphological and cytological analyses revealed increased leaf length-to-width ratios and decreased surface area post-irradiation, characterized by disordered palisade and spongy tissue arrangement and a significant reduction in cell roundness. Transcriptomic analysis identified numerous differentially expressed genes enriched in cell wall metabolism and adaxial-abaxial polarity establishment pathways. Both the abaxial determinant PeKAN2 and the adaxial determinant PeAS1 were significantly downregulated post-irradiation. The synergistic downregulation of PeAS1 and PeKAN2 disrupted the dynamic balance between adaxial and abaxial determinants, driving abnormal anisotropic cell expansion and ultimately altering leaf polar growth. Virus-induced gene silencing of PeKAN2 or PeAS1 individually resulted in significant changes in Phalaenopsis leaf morphology and caused cascading dysregulation of the entire polarity gene network, including PeHD-ZIP, PeYABBY, and PeARF. This study provides new insights into leaf polarity regulation in non-model plants and establishes a theoretical foundation and molecular targets for radiation mutagenesis breeding in orchids.