Comparative genomics of the RBOH gene family across 22 Brassicaceae species and characterization of BnRBOHs

Abstract Respiratory burst oxidase homologs (RBOHs) are key enzymes on the plant plasma membrane that mediate stress-induced extracellular reactive oxygen species (ROS) bursts, playing important roles in plant growth, development, and stress responses. Previous studies on the RBOH gene family have largely focused on identification and functional analysis in single species, and systematic phylogenetic analyses across a broad range of Brassicaceae species remain lacking. In this study, based on genomic data from 22 Brassicaceae species, we systematically identified the RBOH gene family and obtained a total of 314 RBOH genes. Phylogenetic analysis classified these 314 RBOH genes into five conserved groups. Whole genome duplication (WGD) was the major driving force for gene family expansion, contributing 58.6% of the genes (184 genes). Synteny analysis revealed the evolutionary and retention patterns of RBOH genes between Brassica napus and its diploid progenitors Brassica rapa and Brassica oleracea. Promoter analysis revealed abundant stress-responsive and hormone-responsive cis-elements, with the abscisic acid-responsive element ABRE and the hormone cross-regulatory element as-1 being the most enriched. Expression analysis showed that BnRBOH genes exhibited differential response patterns under abiotic stresses such as drought and salt stress, with significantly more responses in root tissues than in leaf tissues. Transcription factor regulatory network analysis identified 244 transcription factors regulating 18 BnRBOH genes, with stress-responsive core transcription factor families such as ERF, NAC, and WRKY being dominant. This study fills the gap in large-scale phylogenetic research on the RBOH gene family in Brassicaceae and provides priority candidate targets — root-specific, stress-inducible BnRBOH genes — for stress tolerance improvement in rapeseed.