Redox buffering and HO orchestrate the vegetative development of Marchantia polymorpha.

Redox processes and reactive oxygen species (ROS) signaling play not only a crucial role in stress responses but also in angiosperm development. However, the specific mechanisms by which redox homeostasis regulates meristems and growth in non-vascular plants remain poorly understood. Here, we demonstrate the applicability of the roGFP2-hGrx1 and HyPer7 redox-biosensors for imaging dynamic glutathione (GSH) and HO redox states in the liverwort Marchantia polymorpha. RoGFP2-hGrx1 microscopy, together with analysis of knockdown plants of the GAMMA GLUTAMYLCYSTEINE SYNTHETASE gene MpGSH1, unveiled a more reduced GSH redox potential (E) in the meristematic region and a more oxidized state in differentiated thallus tissues. Rather than absolute E values, maintenance of a GSH redox gradient is crucial for proper vegetative development. High-resolution HyPer7 analysis detected a heterogenous HO accumulation. Overall, the meristematic region exhibits lower HO levels. Notably, a small zone with higher sensor oxidation is localized in the center of the meristem, likely comprising stem cells and proliferating derivatives. In differentiated thallus tissue, higher levels of HO were detected. External HO application revealed dose-dependent effects that promote or arrest growth. Overproliferation in the meristematic region, driven by treatment with the CLAVATA3/EMBRYO SURROUNDING REGION-related peptide (MpCLE2p) increased HO levels in expanded meristems, supporting the importance of HO signaling in balancing cell proliferation and differentiation in M. polymorpha. Further comparative high-resolution redox sensor studies in bryophytes and vascular plants can shed light on the contribution of redox processes to the regulation of developmental processes and the formation of increasingly complex land plants.