Wound Induces Multi-Layered Barrier Formation in Mature Leaves via Phytohormone Signaling and ATML1-Mediated Epidermal Specification

The epidermis of plants forms a protective barrier against biotic and abiotic stress. Little is known about how breaches in the epidermis are repaired, especially those of mature leaves. Here, we investigated wound healing in the mature leaves of Arabidopsis. We discover a novel wound protection mechanism comprising a multi-layered ligno-suberized barrier covered with cuticular wax. This barrier is formed by mesophyll cells that adopt an epidermal fate. This cell fate transition is regulated in two steps by ATML1, a key transcription factor in epidermal specification. First, mesophyll cells of protective layer 1 (P1), just beneath the wound, transition into epidermal cells, sealing the wound by depositing cuticle, a mechanism that involves signaling through ethylene and reactive oxygen species produced by RBOHE. This signaling also promotes P1 cell death, ensuring wax accumulation on the surface. Second, the underlying protective layer 2 (P2) undergoes ligno-suberization, driven by jasmonic acid and RBOHD, forming a cork-like layer on the leaf surface. ATML1 regulates this process in P2 as well. Thus, wound healing in mature leaves involves integration of ethylene and jasmonic acid signaling with ATML1-mediated epidermal cell specification to coordinate cell layer-specific functions, including cuticular wax formation and ligno-suberization. This novel protective mechanism also occurs in the leaves of tobacco and Capsella, suggesting it is widespread. Overall design: RNA-sequencing of 4-week-old 8th Arabidopsis leaf given mechanical wound