Real‐time monitoring of subcellular H2O2 dynamics by genetically encoded probe roGFP2‐PRXIIB

In plants, genetically encoded probes based on redox-sensitive green fluorescent protein (roGFP) have been used to detect hydrogen peroxide (H₂O₂) levels by fusing exogenous thiol peroxidases, such as Orp1 and Tsa2. However, the effectiveness of these thiol peroxidases compared to endogenous ones remains unexplored. Here, we develop a H₂O₂ probe by fusing roGFP2 to an endogenous H₂O₂ sensor, type II peroxiredoxin (PRXIIB), which displayed enhanced responsiveness and conversion kinetics compared to roGFP2-Orp1 in vitro and superior sensitivity to H₂O₂ in vivo. The roGFP2-PRXIIB probe allowed robust visualization of H₂O₂ production in abiotic and biotic stresses, and growing pollen tubes. We further targeted roGFP2-PRXIIB to cytosol, nuclei, mitochondria and chloroplasts to monitor H₂O₂ accumulation in real time in different subcellular compartments during immune activation, and the analyses revealed different temporal patterns of H₂O₂ accumulation during pattern- and effector-triggered immune responses in different compartments. Taken together, the work provides an ultra-sensitive probe for H₂O₂ dynamics in diverse plant biological processes.