J Integr Plant Biol ›› 2025, Vol. 67 ›› Issue (8): 2044-2057.DOI: 10.1111/jipb.13938

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  • 收稿日期:2025-02-27 接受日期:2025-04-30 出版日期:2025-08-01 发布日期:2025-08-05

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

Man Hu1, Yu Liang2,3, Jiang‐Guo Meng4, Kangmin He2,3, Wei‐Cai Yang2,5, Guozhi Bi6* and Jian‐Min Zhou1,5*   

  1. 1. State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, the Chinese Academy of Sciences, Beijing 100101, China
    2. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, the Chinese Academy of Sciences, Beijing 100101, China
    3. University of Chinese Academy of Sciences, Beijing 100049, China
    4. School of Life Sciences, Southwest University, Chongqing 400715, China
    5. Yazhouwan National Laboratory, Sanya 572024, China
    6. State Key Laboratory of Plant Environmental Resilience, College of Biological Sciences, China Agricultural University, Beijing 100193, China

    *Correspondences: Guozhi Bi (guozhibi@cau.edu.cn); Jian‐Min Zhou (jmzhou@genetics.ac.cn, Dr. Zhou is fully responsible for the distribution of all materials associated with this article)
  • Received:2025-02-27 Accepted:2025-04-30 Online:2025-08-01 Published:2025-08-05
  • Supported by:
    The work was supported by the National Natural Science Foundation of China (32330056 and 32120103004 to J.‐M.Z., 32170288 to G.B.).

Abstract: 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.

Key words: abiotic stress, H2O2, plant immunity, roGFP2‐PRXIIB, subcellular compartments

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