HyPer7 imaging Unveils the role of hydrogen peroxide in Arabidopsis apical hook development

The apical hook in Arabidopsis plays a critical role in safeguarding the shoot apical meristem during seedling emergence from soil. This study elucidates the dynamic regulation of hydrogen peroxide (H₂O₂) in hook development through pharmacological interventions and the HyPer7 biosensor. Optical microscopy revealed that 18 mM aminoacetonitrile (AAN) induced complete hook opening (∼25° angle) at 36 h post-germination by elevating H₂O₂ levels, whereas 2 μM diphenyleneiodonium chloride (DPI), 10 mM dimethylthiourea (DMTU), 5 mM DMTU, and 10 mM potassium iodide (KI) suppressed opening, maintaining angles of 174°, 227°, 150°, and 130°, respectively, through H₂O₂ depletion. HyPer7-based imaging demonstrated a pronounced H₂O₂ gradient at 36 h, with higher levels in the concave side (fluorescence ratio 1.8 vs. 1.2 in the convex side). This gradient equilibrated by 84 h (1.5 vs. 1.4), coinciding with hook maturation. Our findings establish H₂O₂ as a spatiotemporal regulator of asymmetric cell growth during hook morphogenesis and validate HyPer7 as a precision tool for ROS dynamics in plant developmental studies.