Abstract text: Apoplastic barriers, such as the endodermis and phellem, are essential for root tissue compartmentalization and protection against environmental stress. Lignin and suberin are hydrophobic polymers that contribute to barrier function. While lignin is also present in suberized tissues, the spatiotemporal deposition patterns of lignin and its relationship with suberin remain unclear. The mature primary root of 15-day-old Arabidopsis plants provides an ideal system to simultaneously study the development of two ligno-suberized barriers: the endodermis and the phellem. Whereas the endodermis predominates in young root regions, the phellem progressively replaces it toward the hypocotyl. Using gene expression markers, histochemical staining, and confocal microscopy with polymer-specific dyes, we mapped and followed the progression of lignin and suberin deposition. Electron microscopy provided ultrastructural and topochemical information on ligno-suberized cell walls. Our results reveal distinct spatiotemporal patterns and identify two separate polyaromatic/lignin domains in suberized cells. Analyses of suberin-deficient mutants validate the ligno-suberization model and shed light on the macromolecular structure of the ligno-suberized cell wall. Overall, our findings establish how lignin and suberin coordinate to reinforce apoplastic barriers, providing essential insight into barrier formation and function.