CaRcd1 positively regulates pepper (Capsicum annuum L.) regeneration by interacting with the transcription factor CaGATA26 to inhibiting CaTMKL1 expression

Pepper serves as a significant vegetable and spice crop, with fruits abundant in bioactive compounds and substantial potential applications in industries. As the crop widely regarded as recalcitrant to regeneration, the regeneration and transgenic system based on tissue culture for pepper remains underdeveloped. This study initially evaluated 60 varieties for regeneration potential and identified the differentiation of normal shoots (with meristem) during redifferentiation, as the most critical limiting factor in pepper regeneration. Selected R60 as the variety with superior regeneration capacity through pharmacological and RNA-seq analyses of the typical features of dedifferentiation and redifferentiation. Within the same genetic context, we observed that CaRcd1 ( Required for cell differentiation 1 ) was positively associated with the regenerative ability of pepper, while CaTMKL1 ( Transmembrane Kinase-Like 1 ) was negatively associated. Furthermore, transgenic experiments demonstrated that CaRcd1 -OE significantly doubled the rate of normal shoot differentiation. At the protein level, transcription factor CaGATA26, as an interacting protein, is screened by IP-MS, and the interaction could inhibit the transcriptional promotion of TF CaGATA26 on the auxin binding protein encoding gene CaTMKL1 , thereby reducing the expression of CaTMKL1 , inhibiting the transfer of auxin in callus can improve the regenerative ability of pepper. Our study presents a novel pathway for regulating regeneration ability, centered on CaRcd1, and provides a novel reference framework for tissue culture in plants facing regeneration challenges. • Selected R60 as the most regenerative pepper variety, and improved the pepper regeneration efficiency. • Improved the transgenic system of pepper, and double the rate of normal shoot differentiation by overexpression of CaRcd1 . • Confirmed that CaRcd1 as the core protein positively regulates the shoot differentiation during pepper regeneration by RNA-seq. • Verified the interaction between CaRcd1 monomer and CaRcd1-CaGATA26 complex with CaTMKL1 pro by biochemical experiments. • Confirmed that CaRcd1 regulates pepper regeneration by interacting with CaGATA26 to CaTMKL1 expression.