A full-length GRAS domain protein underpins efficient shoot regeneration in pepper

Pepper (Capsicum annuum L.) is a recalcitrant species regarding shoot regeneration, a trait that serves as a major bottleneck for the application of genetic engineering tools. In this study, comparative genetic analysis between a rare high-regeneration cultivar and a common low-efficiency cultivar identified a single nucleotide polymorphism (SNP) in PHYTOCHROME A SIGNAL TRANSDUCTION 1 (CaPAT1) that determines shoot regeneration efficiency. The T478C SNP in the high-efficiency cultivar converts a stop codon into an Arg codon, leading to translational read-through into the neighboring gene and forming an intact GRAS domain. This SNP-mediated formation of full-length CaPAT1 is essential for its dimerization. Notably, the overexpression of CaPAT1T478C in multiple low-efficiency cultivars, including both hot and bell peppers, significantly improved both shoot regeneration and transformation efficiency in the transformed T0 generation. These findings demonstrate the pivotal role of CaPAT1 in enhancing shoot regeneration and provide a robust strategy to overcome recalcitrance in pepper.