| Summary: | Adventitious root (AR) formation is a critical process in cutting propagation of horticultural plants. Brassinosteroids (BRs) have been shown to regulate AR formation in several plant species; however, little is known about their exact effects on pepper AR formation, and the downstream signaling of BRs also remains elusive. In this study, we showed that treatment of 24-Epibrassinolide (EBL, an active BR) at the concentrations of 20–100 nM promoted AR formation in pepper (<i>Capsicum annuum</i>). Furthermore, we investigated the roles of apoplastic reactive oxygen species (ROS), including hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) and superoxide radical (O<sub>2</sub><sup>•−</sup>), in EBL-promoted AR formation, by using physiological, histochemical, bioinformatic, and biochemical approaches. EBL promoted AR formation by modulating cell-wall-located polyamine oxidase (PAO)-dependent H<sub>2</sub>O<sub>2</sub> production and respiratory burst oxidase homologue (RBOH)-dependent O<sub>2</sub><sup>•−</sup> production, respectively. Screening of <i>CaPAO</i> and <i>CaRBOH</i> gene families combined with gene expression analysis suggested that EBL-promoted AR formation correlated with the upregulation of <i>CaPAO1</i>, <i>CaRBOH2</i>, <i>CaRBOH5</i>, and <i>CaRBOH6</i> in the AR zone. Transient expression analysis confirmed that CaPAO1 was able to produce H<sub>2</sub>O<sub>2</sub>, and CaRBOH2, CaRBOH5, and CaRBOH6 were capable of producing O<sub>2</sub><sup>•−</sup>. The silencing of <i>CaPAO1</i>, <i>CaRBOH2</i>, <i>CaRBOH5</i>, and <i>CaRBOH6</i> in pepper decreased the ROS accumulation and abolished the EBL-induced AR formation. Overall, these results uncover one of the regulatory pathways for BR-regulated AR formation, and extend our knowledge of the functions of BRs and of the BRs-ROS crosstalk in plant development.
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