Clathrin‐mediated endocytosis regulates root endodermal suberization via <scp>ROS</scp>

Endomembrane trafficking (ET) plays a crucial role in plant adaptation to environmental stresses, yet its involvement in endodermal root suberization remains poorly understood. Here, we show that disruption of clathrin-mediated endocytosis (CME) or canonical exocytosis led to an ectopic suberin deposition in the Arabidopsis root endodermis toward the root tip. Genetic disruption of endocytosis phenocopied the effects of the CME inhibitor ES9-17, while genetic disruption of clathrin-independent endocytosis led to reduced suberization, suggesting distinct, pathway-specific roles in regulating suberin deposition. Ectopic suberization upon CME inhibition required the CIFs-SGN3-SGN1-RBOHF/D signaling axis, independent of abscisic acid. Notably, CME disruption led to the accumulation of RBOHF in the plasma membrane, driving NADPH oxidase-dependent H₂O₂ accumulation in the endodermis. Scavenging H₂O₂ or inhibiting NADPH oxidases abolished ET disruption-induced suberization, while exogenous H₂O₂ promoted it. Conversely, peroxidase activity inhibition reduced basal suberization but failed to suppress ET disruption-induced enhanced suberization, implicating reactive oxygen species (ROS) as a dominant driver. Our findings reveal a dual ET regulatory mechanism: exocytosis inhibition leads to suberization independently of known pathways, while CME impairment acts via RBOHF-mediated ROS to increase suberization on the endodermis. This study reveals that ET can control endodermal root suberization in Arabidopsis, linking membrane trafficking to apoplastic barrier formation through reactive oxygen species.