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. RNA-sequencing of 4-week-old 8th Arabidopsis leaf given mechanical wound

植物表皮（epidermis）是抵御生物与非生物胁迫的保护性屏障。目前学界对表皮破损后的修复机制，尤其是成熟叶片的表皮破损修复，所知甚少。本研究以拟南芥成熟叶片的伤口愈合为研究对象，揭示了一种全新的伤口保护机制：该机制由多层木质栓化屏障构成，屏障表面覆盖角质层蜡质（cuticular wax）。该屏障由获得表皮细胞命运的叶肉细胞（mesophyll cells）形成。这一细胞命运转变由表皮特化关键转录因子ATML1通过两步调控完成：其一，伤口正下方的第一层保护层（P1）的叶肉细胞转化为表皮细胞，通过沉积角质层实现伤口密封，该过程依赖乙烯信号通路以及RBOHE介导产生的活性氧（reactive oxygen species）；同时该信号通路还会促进P1细胞死亡，确保蜡质在表面积累。其二，下层的第二层保护层（P2）发生木质栓化，该过程由茉莉酸（jasmonic acid）信号与RBOHD驱动，在叶片表面形成类木栓层。ATML1同样对P2的这一过程具有调控作用。综上，成熟叶片的伤口愈合过程整合了乙烯与茉莉酸信号通路，以及ATML1介导的表皮细胞特化过程，以此协调不同细胞层的功能，包括角质层蜡质形成与木质栓化。这一全新的保护机制同样存在于烟草（tobacco）和荠属（Capsella）植物的叶片中，表明该机制具有广谱性。本研究对经机械损伤的4周龄拟南芥第8片叶片开展了RNA测序（RNA-sequencing）