CsBZR4-CsRBOH5.1-BR module regulates opening/closure and expansion of cucumber cotyledons

The hydrogen peroxide (H₂O₂) plays diverse roles in physiological processes in plants. However, whether and how H₂O₂ regulates the opening and expansion of cotyledons in plants remains unknown. We demonstrated that CsRBOH5.1-encoded NADPH oxidase controls diurnal cotyledon opening/closure and expansion. Csrboh5.1 mutants exhibited defective cotyledon movements and reduced brassinosteroids (BR) levels, particularly 28-norbrassinolide and typhasterol. Both exogenous H₂O₂ and 24-epibrassinolide (EBR) treatments rescued these phenotypic defects. Molecular analyses revealed BR biosynthesis promotion by H₂O₂ and reciprocal regulation through CsBZR4 transcription factor. EBR upregulated CsBZR4 expression, which directly bound to and activated the CsRBOH5.1 promoter, as confirmed by yeast one-hybrid and dual-luciferase assays. This establishes a self-reinforcing regulatory module: CsRBOH5.1-derived H₂O₂ enhances BR synthesis, while BR-induced CsBZR4 transcriptionally activates CsRBOH5.1 expression. Transient overexpression assays in Csrboh5.1 mutants demonstrated that CsBZR4 did not directly regulate cotyledon opening. Our findings revealed the first plant RBOH-mediated H₂O₂ regulation of cotyledon development and elucidate a novel 'CsBZR4-CsRBOH5.1-BR' feedback mechanism coordinating redox signaling and phytohormone pathways. This module ensures precise control of cotyledon growth dynamics through reciprocal modulation of oxidative metabolism and brassinosteroids synthesis, providing new insights into the integration of redox biology and hormonal regulation in plant organogenesis.