Genome-Wide Identification and Characterization of the Rboh Gene Family in Populus trichocarpa Reveals PtrRbohB Positively Regulates Adventitious Root Development

Adventitious roots (ARs) are essential for clonal vegetative propagation of woody tree species. Extensive studies have focused on phytohormone and transcription factor regulatory networks during AR development in poplar, yet the regulatory roles of reactive oxygen species (ROS) signaling during AR development remain poorly understood. In this study, we performed a genome-wide identification and systematic characterization of the Respiratory Burst Oxidase Homolog (Rboh) gene family in Populus trichocarpa. A total of 10 PtrRbohs were identified and phylogenetically classified into five subfamilies. Expression profiling revealed distinct tissue-specific and stress-responsive expression patterns among PtrRbohs. Functional validation via CRISPR/Cas9-mediated knockout showed that disruption of PtrRbohB significantly reduced AR length, diameter, and lateral root density in poplar. Heterologous overexpression of PtrRbohB in Arabidopsis thaliana enhanced ROS accumulation, confirming its conserved function in ROS generation. Furthermore, knockout of PtrRohB down-regulated the expression of several AR-positive regulators and up-regulated that of AR-negative regulators in poplar. Collectively, this study provides the direct genetic evidence that Rboh regulates AR formation in poplar, and identifies PtrRbohB as a key positive regulator of AR development. These findings enrich our understanding of woody plant AR formation and provide candidate genes for molecular breeding of poplar rooting traits.