Genome-wide identification of GRF transcription factor in Populus: Regulatory analysis of the miR396-PagGRF1/2e-PagWUSa module in adventitious shoot regeneration

Growth-Regulating Factor (GRF) transcription factors are important regulators that promote adventitious shoot formation and improve genetic transformation efficiency in plants. Poplar ( Populus spp.), known for its rapid growth, is extensively applied in forestry improvement and biotechnology. Organ regeneration serves as the foundation for genetic improvement. The regeneration process in poplar is often regarded as being strongly genotype-dependent and characterized by prolonged regeneration periods. Here, we characterized 22 Pag GRF genes in the genome of '84 K' poplar ( Populus alba × P. glandulosa ) and performed a genome-wide survey to characterize their evolutionary relationships and gene features. Of these genes, Pag GRF1/2e was markedly upregulated during adventitious shoot regeneration. Overexpression of Pag GRF1/2e significantly promoted adventitious shoot regeneration. Whereas overexpression of miR396 strongly suppressed it. Further analysis revealed that PagGRF1/2e binds the Pag WUSa (WUSCHEL) promoter to induce its transcription, forming a regulatory module—miR396–PagGRF1/2e–Pag WUSa —that controls adventitious shoot regeneration in poplar. Overall, our study not only identifies Pag GRF1/2e as a positive regulator of shoot regeneration and provide a well-defined genetic module that could serve as a strategic target for improving regenerative and transformation efficiency in woody species. • 22 PagGRF genes were identified in '84 K' poplar. • PagGRF1/2e shows a novel function in promoting adventitious shoot regeneration. • The miR396-PagGRF1/2e-Pag WUSa module controlling adventitious shoot regeneration was revealed. • This study provides a genetic framework for improving adventitious shoot regeneration in woody plants.