Indispensable roles of RBOH-mediated ROS production in development and morphogenesis of Marchantia polymorpha

Reactive oxygen species (ROS) are now well established as essential signaling molecules in eukaryotes. In plants, NADPH oxidases, known as respiratory burst oxidase homologs (RBOHs), are the primary source of signaling ROS. Although ROS have been implicated in plant development, a causal demonstration of the essential role of RBOH-derived ROS in fundamental developmental processes has remained elusive. This gap largely reflects the extensive genetic redundancy of RBOH genes in angiosperm model systems, which often masks essential developmental functions. Here, we exploited the exceptionally low genetic redundancy of the bryophyte Marchantia polymorpha, which possesses only two RBOHs, MpRBOHA and MpRBOHB. By generating a complete null mutant, we uncovered a severe developmental phenotype forming a disorganized cell mass, which demonstrates that MpRBOHs are indispensable for normal morphogenesis. Phenotypic gradation among the single and double mutants, together with complementation analyses, revealed that the two MpRBOHs function partially redundantly in meristem-driven development. Convergent evidence from constitutive knockout, inducible knockdown, and pharmacological inhibition demonstrates that RBOH-derived ROS positively regulate growth by promoting cell proliferation. Conditional knockout analyses further revealed that MpRBOHs are required from early developmental stages to sustain morphogenesis by maintaining cuticle-cell-wall integrity, which is essential for controlling cellular shape, spatial arrangement, and organ differentiation. Transcriptomic analyses showed that the developmental arrest of the MprbohA rbohB mutant is associated with a failure to activate cell-differentiation programs. Together, our findings uncover a previously unrecognized and fundamental role of RBOH-derived ROS in coordinating physical integrity with cell differentiation during plant development.