24-Epibrassinolide Facilitates Adventitious Root Formation by Coordinating Cell-Wall Polyamine Oxidase- and Plasma Membrane Respiratory Burst Oxidase Homologue-Derived Reactive Oxygen Species in Capsicum annuum L.

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 ( Capsicum annuum ). Furthermore, we investigated the roles of apoplastic reactive oxygen species (ROS), including hydrogen peroxide (H 2 O 2 ) and superoxide radical (O 2 •- ), 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 2 O 2 production and respiratory burst oxidase homologue (RBOH)-dependent O 2 •- production, respectively. Screening of CaPAO and CaRBOH gene families combined with gene expression analysis suggested that EBL-promoted AR formation correlated with the upregulation of CaPAO1 , CaRBOH2 , CaRBOH5 , and CaRBOH6 in the AR zone. Transient expression analysis confirmed that CaPAO1 was able to produce H 2 O 2 , and CaRBOH2, CaRBOH5, and CaRBOH6 were capable of producing O 2 •- . The silencing of CaPAO1 , CaRBOH2 , CaRBOH5 , and CaRBOH6 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.